Cells for immunotherapy engineered for targeting antigen present both on immune cells and pathological cells

ABSTRACT

Methods of developing genetically engineered immune cells for immunotherapy, which can be endowed with Chimeric Antigen Receptors targeting an antigen marker that is common to both the pathological cells and said immune cells (ex: CD38, CS1 or CD70) by the fact that the genes encoding said markers are inactivated in said immune cells by a rare cutting endonuclease such as TALEN, Cas9 or argonaute.

FIELD OF THE INVENTION

The present invention relates to methods of developing geneticallyengineered, preferably non-alloreactive, immune cells for immunotherapy,which are endowed with Chimeric Antigen Receptors targeting an antigenmarker that is common to both the pathological cells and the immunecells (ex: CD38).

The method comprises expressing a CAR directed against said antigenmarker and inactivating the genes in the immune cells contributing tothe presence of said antigen marker on the surface of said immune cells.This inactivation is typically performed by using transgenes encodingRNA-guided endonucleases (ex: Cas9/CRISPR), meganucleases, Zinc-fingernucleases or TAL nucleases. The engineered immune cells, preferablyT-cells, direct their immune activity towards malignant, infected cellsor defective immune cells, while avoiding their mutual destruction,auto-stimulation or aggregation. The invention opens the way to standardand affordable adoptive immunotherapy strategies using immune cells fortreating cancer, infections and auto-immune diseases.

BACKGROUND OF THE INVENTION

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific immune cells generated ex vivo, is a promising strategyto treat viral infections and cancer. The T cells used for adoptiveimmunotherapy, for instance, can be generated either by expansion ofantigen-specific T-cells or redirection of T-cells through geneticengineering (Park, Rosenberg et al. 2011).

Novel specificities in T-cells have been successfully generated throughthe genetic transfer of transgenic T-cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity, however, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T cells.CARs have successfully allowed T cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

The current protocol for treatment of patients using adoptiveimmunotherapy is based on autologous cell transfer. In this approach, Tlymphocytes are recovered from patients, genetically modified orselected ex vivo, cultivated in vitro in order to amplify the number ofcells if necessary and finally infused into the patient. In addition tolymphocyte infusion, the host may be manipulated in other ways thatsupport the engraftment of the T cells or their participation in animmune response, for example pre-conditioning (with radiation orchemotherapy) and administration of lymphocyte growth factors (such asIL-2). Each patient receives an individually fabricated treatment, usingthe patient's own lymphocytes (i.e. an autologous therapy). Autologoustherapies face substantial technical and logistic hurdles to practicalapplication, their generation requires expensive dedicated facilitiesand expert personnel, they must be generated in a short time following apatient's diagnosis, and in many cases, pretreatment of the patient hasresulted in degraded immune function, such that the patient'slymphocytes may be poorly functional and present in very low numbers.Because of these hurdles, each patient's autologous cell preparation iseffectively a new product, resulting in substantial variations inefficacy and safety.

Ideally, one would like to use a standardized therapy in whichallogeneic therapeutic cells could be pre-manufactured, characterized indetail, and available for immediate administration to patients. Byallogeneic it is meant that the cells are obtained from individualsbelonging to the same species but are genetically dissimilar. However,the use of allogeneic cells presently has many drawbacks. Inimmune-competent hosts allogeneic cells are rapidly rejected, a processtermed host versus graft rejection (HvG), and this substantially limitsthe efficacy of the transferred cells. In immune-incompetent hosts,allogeneic cells are able to engraft, but their endogenous T-cellreceptors (TCR) specificities may recognize the host tissue as foreign,resulting in graft versus host disease (GvHD), which can lead to serioustissue damage and death.

In order to provide allogeneic T-cells, the inventors previouslydisclosed a method to genetically engineer T-Cells, in which differenteffector genes, in particular those encoding T-cell receptors, wereinactivated by using specific TAL-nucleases, better known under thetrade mark TALEN™ (Cellectis, 8, rue de la Croix Jarry, 75013 PARIS).This method has proven to be highly efficiency in primary cells usingRNA transfection as part of a platform allowing the mass production ofallogeneic T-cells (WO 2013/176915).

CD38 (cluster of differentiation 38), also known as cyclic ADP ribosehydrolase is a glycoprotein found on the surface of many immune cells(white blood cells), in particular T-cells, including CD4+, CD8+, Blymphocytes and natural killer cells. CD38 also functions in celladhesion, signal transduction and calcium signaling. Structuralinformation about this protein can be found in the UniProtKB/Swiss-Protdatabase under reference P28907. In humans, the CD38 protein is encodedby the CD38 gene which located on chromosome 4. CD38 is amultifunctional ectoenzyme that catalyzes the synthesis and hydrolysisof cyclic ADP-ribose (cADPR) from NAD+ to ADP-ribose. These reactionproducts are deemed essential for the regulation of intracellular Ca2+.Also, loss of CD38 function was associated with impaired immuneresponses and metabolic disturbances (Malavasi F., et al. (2008).“Evolution and function of the ADP ribosyl cyclase/CD38 gene family inphysiology and pathology”. Physiol. Rev. 88(3): 841-86).

On another hand, CD38 protein is a marker of HIV infection, leukemias,myelomas, solid tumors, type II diabetes mellitus and bone metabolism,as well as some other genetically determined conditions. In particular,it has been used as a prognostic marker in leukemia (Ibrahim, S. et al.(2001) CD38 expression as an important prognostic factor in B-cellchronic lymphocytic leukemia. Blood 98:181-186).

Although, cells expressing CD38, as well as many other tumor antigenmarkers referred to in Table 1, such as CD70 and CS1 could be regardedas attractive targets for CARs, the fact that such antigen markers arealso expressed at the surface of most T-cells, has hamperedsignificantly the selection of these markers to perform immunotherapy.

The inventors here provide strategies for immunotherapy involvingpathological cells expressing specific antigen markers also present atthe surface of T-cells, like for instance malignant CD38 positiveB-cells causing leukemia, CD70 and CS1.

SUMMARY OF THE INVENTION

The present invention discloses methods to engineer T-cells intended totarget pathological cells, whereas said pathological cells express oneor several antigen markers that are also present on the surface ofT-cells. Examples of such antigen markers are found in Table 1. Anexample of such antigen marker is CD38. Other examples are CD70 and CS1.By antigen marker is meant the whole protein of an immune-reactivefragment thereof.

According to the invention, the T-cells are engineered in order toinactivate the expression of the genes encoding such antigen markers, orinvolved into the presentation of such antigen marker on the cellsurface.

This inactivation is preferably performed by a genome modification, moreparticularly through the expression in the T-cell of a specificrare-cutting endonuclease able to target a genetic locus directly orindirectly involved in the production or presentation of said antigenmarker at the surface of the T-cell. Different types of rare-cuttingendonucleases can be used, such as Meganucleases, TAL-nucleases,zing-finger nucleases (ZFN), or RNA/DNA guided endonucleases likeCas9/CRISPR or Argonaute.

According to a preferred embodiment, the T-cells are endowed with atleast one chimeric antigen receptors (CAR) allowing a specific bindingof said cells bearing said targeted antigen marker.

According to another embodiment, the T-cells can be further engineeredto make them allogeneic, especially by deleting genes involved intoself-recognition, such as those, for instance, encoding components ofT-cell receptors (TCR) or HLA complex.

The present invention encompasses the isolated cells or cell linescomprising the genetic modifications set forth in the detaileddescription, examples and figures, as well as any of the proteins,polypeptides or vectors useful to engineer said T-cells.

As a result of the invention, the engineered T-cells can be used astherapeutic products, ideally as an “off the shelf” product, in methodsfor treating or preventing cancer, infections or auto-immune disease.

Preferred immune cells according to the present invention are thoseresulting into the phenotypes:

-   -   [CAR targeting a antigen marker of Table1]⁺[antigen marker of        Table1]⁻ such as the following ones:    -   [CAR CD38]⁺[CD38]⁻, preferably also [TCR] negative;    -   [CAR CD70]⁺[CD70]⁻, preferably also [TCR] negative;    -   [CAR CS1]⁺[CS1]⁻, preferably also [TCR] negative;        for their use as therapeutic products, preferably allogeneic        ones.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1: Schematic representation of an engineered T-cell according tothe present invention disrupted for CD38 and endowed with a chimericantigen receptor (represented as a single-chain CAR) targeting amalignant cell bearing the antigen marker CD38.

FIG. 2: Schematic representation of a multi-subunit chimeric antigenreceptor.

FIG. 3: Schematic representation of a therapeutic strategy according tothe invention combining T-cells endowed with a multi-subunit CAR andcirculating bi-specific antibody. In this particular aspect, thereceptor present on the extracellular chain of the multi-subunit CAR iscomposed of an epitope which is recognized by a bi-specific antibody.The bi-specific antibody is intended to bind said epitope one the onehand and the antigen marker on the other hand to facilitate the bindingof the T-cell to the pathological cell.

FIG. 4: Schematic representation of a therapeutic strategy according tothe invention combining T-cells endowed with a multi-subunit CAR andcirculating monoclonal antibody. In this particular aspect, the receptorpresent on the extracellular chain of the multi-subunit CAR is composed,for instance, of a Fc receptor intended to bind a monoclonal antibodythat is directed against the antigen marker. The monoclonal antibodyincreases the chance of T-cells binding the pathological cells.

FIG. 5: Schematic representation of a therapeutic strategy according tothe invention combining T-cells endowed with a multi-subunit CAR thatcomprises two extracellular cellular domains and one circulatingbi-specific antibody. In this particular aspect, the extracellularcellular domains are located on distinct sub-units. These domains arerespectively composed of an epitope that is recognized by a bi-specificantibody and of a receptor targeting an antigen. The receptor isdirected against a first antigen marker, whereas the bi-specificantibody is intended to bind the epitope and a second antigen marker.This display aims to selectively target pathological cells bearing attheir surface both the first and second antigen markers.

FIG. 6: display is similar to FIG. 5, but stimulation and co-stimulationdomains (respectively 4-1BB and CD3zeta protein domains) have beenexchanged to modulate the intensity of the activation of the T-cellresulting from the binding of the chimeric antigen receptor with thepathological cell.

FIG. 7: display is similar to FIG. 5, but stimulation and co-stimulationdomains (respectively 4-1BB and CD3zeta protein domains) have beenexchanged and one CD3zeta domain has been added to increase theintensity of the activation of the T-cell resulting from the binding ofthe chimeric antigen receptor with the pathological cell.

FIG. 8: Schematic representation of a therapeutic strategy according tothe invention combining T-cells endowed with a multi-subunit CAR thatcomprises two extracellular cellular domains and one circulatingmonoclonal antibody. In this particular aspect, the extracellularcellular domains are located on distinct sub-units. These domains arerespectively composed of an antigen binding domain targeting an antigenmarker and a Fc receptor intended to bind a monoclonal antibody that isdirected against a second antigen marker. This display aims toselectively target pathological cells bearing at their surface both thefirst and second antigen markers.

FIG. 9: CD38 expression by activated T cells. A. CD38 expression by Tcells at day 6 after activation with CD3/CD28 coated beads+IL2. B.Longitudinal analysis of CD38 expression by T cells during 17 days afteractivation.

FIG. 10 Knock-out (KO) on CD38 gene: A. Position on CD38 exon 1 sequenceof the 3 differents TALEN (T2, T4 and T5) designed to knock out Cd38 inT cell. B. Expression of CD38 in T cells after transfection with theTALEN CD38ex1_T2. C. CD38 staining to control for the purification ofCD38 KO T cells.

FIG. 11: CD38 CAR: A. Representation of the 3 versions of CARs designed.B. CD38 expression level by the target cell lines.

FIG. 12: Timing experiment for the engineering of the CAR CS1+ and KOCS1 T-cells and their subsequent testing;

FIG. 13: Constructs of T01, T02 and T03 with the TAL repeats used forthe KO of CS1 gene;

FIG. 14: Target location for the TALs T01, T02 and T03 within the CS1(SLAM F7) gene. T01 and T02 target the exon 1 (FIG. 14A), whereas T03targets the exon 2 (FIG. 14B).

FIG. 15A: Measurement of percentage of target cell viability for TALEnor not TALEn transfected combined with CAR+ or not transduced cells: areduced cell viability of CS1(+) cells shown when they were co-culturedwith CAR+ T-cells, while no impact on CS1(−) cell viability wasobserved.

FIG. 15B: Measurement of percentage of specific cell lysis (CS1+)calculated using the flow cytometry data. It is shown that specific celllysis is 2-times higher when T-cells have been transfected with TALEntargeting the CS1 gene prior to CAR transduction.

FIG. 16: Results of FACS analysis from cytoxic activity experiment,which show that transduction efficiencies are higher in mock transfectedcells than in cells that have been transfected with TALEn targeting theCS1 gene (NTD: not transduced).

FIG. 17: Results from FACS analysis when the different samples arereactivated with CD3/CD28 beads at D11 after transduction, showing thetransduction efficiencies and CD8/CS1 expression levels in each sample.An increase in CS1 levels upon re-activation is observed in mocktransfected cells, while a low amount of cells are able to express CS1in the TALEn transfected populations.

Table 1: Different cytopulse programs used for T-cells electroporation.

Table 2: appropriate target sequences for the guide RNA using Cas9 inT-cells Table 3: List of genes encoding immune checkpoint proteins Table4: Cluster of differentiation (CD) antigen markers found to be expressedon the surface of T-cells, while being characteristic of different typesof tumors.

Table 5 to 13: Main surface antigen markers expressed in T-cells, whilebeing over-expressed in solid tumor cells from various types of cancer.The listed antigen markers were identified as explained in Example 1.

Table 5: colon tumor cells;

Table 6: breast tumor cells;

Table 7: digestive track tumor cells;

Table 8: kidney tumor cells;

Table 9: liver tumor cells;

Table 10: lung tumor cells;

Table 11: ovary tumor cells;

Table 12: pancreas tumor cells;

Table 13: prostate tumor cells;

Table 14: Main surface antigen markers expressed in T-cells, while beingover-expressed in liquid tumor cells from various types of cancer (ALL,AML, CML, MDS, CLL, CTRL). The listed antigen markers were identified asexplained in Example 1.

Table 15: Sequences of the tested CD38 target and TALENs forinactivation of the CD38 antigen;

Table 16: Sequences of two other CD38 targets and the correspondingTALENs for their inactivation;

Table 17: Sequences of VH and VL chains of the scFv anti-CD38 antibodiesdaratumumab and MOR202 and of specific CDRs for VH and VL chains

Table 18: Polypeptide sequence of the 3 different structures of scFvdaratumumab-based anti-CD38 CARs and of the individual components used;

Table 19: Sequences of VH and VL chains of the scFv anti-CS1 antibodies;

Table 20: Polypeptide sequence of anti-CS1 CARs based on the V1, V2 andV3 versions in FIG. 11A;

Table 21: Sequences of the CS1 target and TALENs for its inactivation;

Table 22: Sequences of the CD70 target and TALENs for its inactivation;

Table 23: Polynucleotide and nucleic acid sequences of VH and VL chainsof the scFv anti-CD70 Ab4, Ab8 and 1F6 antibodies;

Table 24: Polypeptide sequence of anti-CD70 CARs based on the V1, V2 andV3 versions in FIG. 11A

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific termsused have the same meaning as commonly understood by a skilled artisanin the fields of gene therapy, biochemistry, genetics, and molecularbiology.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson Inc, Library of Congress, USA); Molecular Cloning: A LaboratoryManual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J.Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Harries & S. J. Higgins eds. 1984); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture OfAnimal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelsonand M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

In a general aspect, the present invention relates to methods for newadoptive immunotherapy strategies in treating diseases linked with thedevelopment of pathological cells, such as cancer, infections andauto-immune diseases.

As a main objective of the invention is the possibility to targetpathological cells that bear specific antigen markers in common withT-cells. By pathological cell is meant any types of cells present in apatient, which are deemed causing health deterioration.

In general, pathological cells are malignant or infected cells that needto be reduced or eliminated to obtain remission of a patient.

In a first embodiment, the method of the invention concerns a method ofpreparing appropriate immune cells, preferably T-cells for immunotherapycomprising the step of:

-   -   (a) Genetically inactivating or mutating a gene in an immune        cell, which is involved in the expression or presentation of an        antigen marker, said antigen marker being known to be present        both on the surface of said T-cell and the pathological cell;    -   (b) Expressing into said immune cell a transgene encoding a        chimeric antigen receptor directed against said antigen marker        present at the surface of said pathological cell.

The immune cells according to the invention are endowed with a chimericantigen receptor directed to an antigen marker that is commonlyexpressed by the pathological cells and immune cells, or known to bepresent on the surface of said T Cells. The expression “known to bepresent” means that the antigen marker is reported to be found on thesurface of the immune cells grown in natural conditions in-vivo,especially in the blood, but not necessarily when they are culturedin-vitro. In any event, the method of the invention results into theabsence of the antigen marker on the surface of the immune cell, therebypreventing the chimeric antigen receptor from reacting with theengineered T-cell surface. In this respect, the method may include afurther step of purifying the resulting T-cells by excluding the cellspresenting said marker antigen on their surface.

As shown in Table 4, this invention relates to an important number ofantigen marker candidates reported to be expressed by tumor cells, butalso by T-cells. Some of them, like CD38, have been used as specificmarkers in diagnostic methods for a while, especially with respect toLeukemia pathological cells, but not in therapy. Indeed, although thesemarkers were identified in the art as quite specific markers, they couldnot be used as targets for immunotherapy because antibodies directedagainst these markers would have destroyed or interfered with patients'T-cells. The present inventors have established that CS1 and CD70 arealso present on the surface of T-cells and that expressing CARstargeting CS1 and CD70 in such T cells leads to their depletion (seeexample 2).

According to a preferred embodiment of the invention, the gene mutationor inactivation of step a) of the above method is performed using arare-cutting endonuclease.

By inactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form. In particular embodiments, thegenetic modification of the method relies on the expression, in providedcells to engineer, of a rare-cutting endonuclease such that samecatalyzes cleavage in one targeted gene thereby inactivating saidtargeted gene. The nucleic acid strand breaks caused by the endonucleaseare commonly repaired through the distinct mechanisms of homologousrecombination or non-homologous end joining (NHEJ). However, NHEJ is animperfect repair process that often results in changes to the DNAsequence at the site of the cleavage. Mechanisms involve rejoining ofwhat remains of the two DNA ends through direct re-ligation (Critchlowand Jackson 1998) or via the so-called microhomology-mediated endjoining (Betts, Brenchley et al. 2003; Ma, Kim et al. 2003). Repair vianon-homologous end joining (NHEJ) often results in small insertions ordeletions and can be used for the creation of specific gene knockouts.Said modification may be a substitution, deletion, or addition of atleast one nucleotide. Cells in which a cleavage-induced mutagenesisevent, i.e. a mutagenesis event consecutive to an NHEJ event, hasoccurred can be identified and/or selected by well-known method in theart.

The term “rare-cutting endonuclease” refers to a wild type or variantenzyme capable of catalyzing the hydrolysis (cleavage) of bonds betweennucleic acids within a DNA or RNA molecule, preferably a DNA molecule.Particularly, said nuclease can be an endonuclease, more preferably arare-cutting endonuclease which is highly specific, recognizing nucleicacid target sites ranging from 10 to 45 base pairs (bp) in length,usually ranging from 10 to 35 base pairs in length, more usually from 12to 20 base pairs. The endonuclease according to the present inventionrecognizes at specific polynucleotide sequences, further referred to as“target sequence” and cleaves nucleic acid inside these target sequencesor into sequences adjacent thereto, depending on the molecular structureof said endonuclease. The rare-cutting endonuclease can recognize andgenerate a single- or double-strand break at specific polynucleotidessequences.

In a particular embodiment, said rare-cutting endonuclease according tothe present invention is a RNA-guided endonuclease such as theCas9/CRISPR complex. RNA guided endonucleases constitute a newgeneration of genome engineering tool where an endonuclease associateswith a RNA molecule. In this system, the RNA molecule nucleotidesequence determines the target specificity and activates theendonuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al.2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013).

Cas 9

Cas9, also named Csn1 (COG3513) is a large protein that participates inboth crRNA biogenesis and in the destruction of invading DNA. Cas9 hasbeen described in different bacterial species such as S. thermophiles,Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski etal. 2012) and S. Pyogenes (Deltcheva, Chylinski et al. 2011). The largeCas9 protein (>1200 amino acids) contains two predicted nucleasedomains, namely HNH (McrA-like) nuclease domain that is located in themiddle of the protein and a splitted RuvC-like nuclease domain (RNase Hfold) (Makarova, Grishin et al. (2006).

By “Cas9” is meant an engineered endonuclease or a homologue of Cas9which is capable of processing target nucleic acid sequence. Inparticular embodiment, Cas9 can induce a cleavage in the nucleic acidtarget sequence which can correspond to either a double-stranded breakor a single-stranded break. Cas9 variant can be a Cas9 endonuclease thatdoes not naturally exist in nature and that is obtained by proteinengineering or by random mutagenesis. Cas9 variants according to theinvention can for example be obtained by mutations i.e. deletions from,or insertions or substitutions of at least one residue in the amino acidsequence of a S. pyogenes Cas9 endonuclease (COG3513). In the frameaspects of the present invention, such Cas9 variants remain functional,i.e. they retain the capacity of processing a target nucleic acidsequence. Cas9 variant can also be homologues of S. pyogenes Cas9 whichcan comprise deletions from, or insertions or substitutions of, at leastone residue within the amino acid sequence of S. pyogenes Cas9. Anycombination of deletion, insertion, and substitution may also be made toarrive at the final construct, provided that the final constructpossesses the desired activity, in particular the capacity of binding aguide RNA or nucleic acid target sequence.

RuvC/RNaseH motif includes proteins that show wide spectra ofnucleolytic functions, acting both on RNA and DNA (RNaseH, RuvC, DNAtransposases and retroviral integrases and PIWI domain of Argonautproteins). In the present invention the RuvC catalytic domain of theCas9 protein can be characterized by the sequence motif:D-[I/L]-G-X-X-S-X-G-W-A, wherein X represents any one of the natural 20amino acids and [I/L] represents isoleucine or leucine. In other terms,the present invention relates to Cas9 variant which comprises at leastD-[I/L]-G-X-X-S-X-G-W-A sequence, wherein X represents any one of thenatural 20 amino acids and [I/L] represents isoleucine or leucine.

HNH motif is characteristic of many nucleases that act ondouble-stranded DNA including colicins, restriction enzymes and homingendonucleases. The domain HNH (SMART ID: SM00507, SCOP nomenclature:HNHfamily) is associated with a range of DNA binding proteins, performing avariety of binding and cutting functions. The ones with known functionare involved in a range of cellular processes including bacterialtoxicity, homing functions in groups I and II introns and inteins,recombination, developmentally controlled DNA rearrangement, phagepackaging, and restriction endonuclease activity (Dalgaard, Klar et al.1997). These proteins are found in viruses, archaebacteria, eubacteria,and eukaryotes. Interestingly, as with the LAGLI-DADG and the GIY-YIGmotifs, the HNH motif is often associated with endonuclease domains ofself-propagating elements like inteins, Group I, and Group II introns(Dalgaard, Klar et al. 1997). The HNH domain can be characterized by thepresence of a conserved Asp/His residue flanked by conserved His(amino-terminal) and His/Asp/Glu (carboxy-terminal) residues at somedistance. A substantial number of these proteins can also have a CX2Cmotif on either side of the central Asp/His residue. Structurally, theHNH motif appears as a central hairpin of twisted β-strands, which areflanked on each side by an a helix (Kleanthous, Kuhlmann et al. 1999).The large HNH domain of Cas9 is represented by SEQ ID NO. 5. In thepresent invention, the HNH motif can be characterized by the sequencemotif: Y-X-X-D-H-X-X-P-X-S-X-X-X-D-X-S, wherein X represents any one ofthe natural 20 amino acids. The present invention relates to a Cas9variant which comprises at least Y-X-X-D-H-X-X-P-X-S-X-X-X-D-X-Ssequence wherein X represents any one of the natural 20 amino acids.

This invention can be of particular interest to easily do targetedmultiplex gene modifications and to create an inducible nuclease systemby introduction of the guide RNA to the Cas9 cells. For the purpose ofthe present invention, the inventors have established that Cas9 proteincan be divided into two separate split Cas9 RuvC and HNH domains whichcan process target nucleic acid sequence together or separately with theguide RNA.

Also the RuvC and HNH domains from different RNA guided endonucleases orCas homologues may be assembled to improve nuclease efficiency orspecificity. The domains from different species can be either split intotwo proteins or fused to each other to form a variant Cas protein. TheCas9 split system is deemed particularly suitable for an induciblemethod of genome targeting and to avoid the potential toxic effect ofthe Cas9 overexpression within the cell. Indeed, a first split Cas9domain can be introduced into the cell, preferably by stablytransforming said cell with a transgene encoding said split domain.Then, the complementary split part of Cas9 can be introduced into thecell, such that the two split parts reassemble into the cell toreconstitute a functional Cas9 protein at the desired time.

The reduction of the size of the split Cas9 compared to wild type Cas9ease the vectorization and the delivery into the cell, for example, byusing cell penetrating peptides. Re-arranging domains from different Casproteins, allows to modulate the specificity and nuclease activity, forinstance, by targeting PAM motifs that are slightly different from S.pyogenes Cas9

Split Cas9 System

The previous characterization of the RuvC and HNH domains has promptedthe inventors to engineer Cas9 protein to create split Cas9 protein.Surprisingly, the inventors showed that these two split Cas9 couldprocess together or separately the nucleic acid target. This observationallows developing a new Cas9 system using split Cas9 protein. Each splitCas9 domains can be prepared and used separately. Thus, this splitsystem displays several advantages for vectorization and delivery of theRNA guided endonuclease in T-cells, allowing delivering a shorter and/orinactive protein, and is particularly suitable to induce genomeengineering in T-cells at the desired time and thus limiting thepotential toxicity of an integrated Cas9 nuclease.

By “Split Cas9” is meant here a reduced or truncated form of a Cas9protein or Cas9 variant, which comprises either a RuvC or HNH domain,but not both of these domains. Such “Split Cas9” can be usedindependently with guide RNA or in a complementary fashion, like forinstance, one Split Cas9 providing a RuvC domain and another providingthe HNH domain. Different split RNA guided endonucleases may be usedtogether having either RuvC and/or NHN domains.

Each Cas9 split domain can be derived from the same or from differentCas9 homologues. Many homologues of Cas9 have been identified in genomedatabases.

Said Cas9 split domains (RuvC and HNH domains) can be simultaneously orsequentially introduced into the cell such that said split Cas9domain(s) process the target nucleic acid sequence in the cell. SaidCas9 split domains and guide RNA can be introduced into the cell byusing cell penetrating peptides or other transfection methods asdescribed elsewhere.

In another aspect of the invention, only one split Cas9 domain, referredto as compact Cas9 is introduced into said cell. Indeed, surprisinglythe inventors showed that the split Cas9 domain comprising the RuvCmotif as described above is capable of cleaving a target nucleic acidsequence independently of split domain comprising the HNH motif. Thus,they could establish that the guideRNA does not need the presence of theHNH domain to bind to the target nucleic acid sequence and issufficiently stable to be bound by the RuvC split domain. In a preferredembodiment, said split Cas9 domain alone is capable of nicking saidtarget nucleic acid sequence.

Each split domain can be fused to at least one active domain in theN-terminal and/or C-terminal end, said active domain can be selectedfrom the group consisting of: nuclease (e.g. endonuclease orexonuclease), polymerase, kinase, phosphatase, methylase, demethylase,acetylase, desacetylase, topoisomerase, integrase, transposase, ligase,helicase, recombinase, transcriptional activator (e.g. VP64, VP16),transcriptional inhibitor (e. g; KRAB), DNA end processing enzyme (e.g.Trex2, Tdt), reporter molecule (e.g. fluorescent proteins, lacZ,luciferase).

HNH domain is responsible for nicking of one strand of the targetdouble-stranded DNA and the RuvC-like RNaseH fold domain is involved innicking of the other strand (comprising the PAM motif) of thedouble-stranded nucleic acid target (Jinek, Chylinski et al. 2012).However, in wild-type Cas9, these two domains result in blunt cleavageof the invasive DNA within the same target sequence (proto-spacer) inthe immediate vicinity of the PAM (Jinek, Chylinski et al. 2012). Cas 9can be a nickase and induces a nick event within different targetsequences.

As non-limiting example, Cas9 or split Cas9 can comprise mutation(s) inthe catalytic residues of either the HNH or RuvC-like domains, to inducea nick event within different target sequences. As non-limiting example,the catalytic residues of the Cas9 protein are those corresponding toamino acids D10, D31, H840, H868, N882 and N891 or aligned positionsusing CLUSTALW method on homologues of Cas Family members. Any of theseresidues can be replaced by any other amino acids, preferably by alanineresidue. Mutation in the catalytic residues means either substitution byanother amino acids, or deletion or addition of amino acids that inducethe inactivation of at least one of the catalytic domain of cas9. (cf.In a particular embodiment, Cas9 or split Cas9 may comprise one orseveral of the above mutations. In another particular embodiment, splitCas9 comprises only one of the two RuvC and HNH catalytic domains. Inthe present invention, Cas9 from different species, Cas9 homologues,Cas9 engineered and functional variant thereof can be used. Theinvention envisions the use of any RNA guided endonuclease or split RNAguided endonucleases variants to perform nucleic acid cleavage in agenetic sequence of interest.

Preferably, the Cas9 variants according to the invention have an aminoacid sequence sharing at least 70%, preferably at least 80%, morepreferably at least 90%, and even more preferably 95% identity with Cas9of S. Pyogenes (COG3513).

Meganucleases

Rare-cutting endonuclease can also be a homing endonuclease, also knownunder the name of meganuclease. Such homing endonucleases are well-knownto the art (Stoddard 2005). Homing endonucleases are highly specific,recognizing DNA target sites ranging from 12 to 45 base pairs (bp) inlength, usually ranging from 14 to 40 bp in length. The homingendonuclease according to the invention may for example correspond to aLAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIGendonuclease. Preferred homing endonuclease according to the presentinvention can be an I-Crel variant. A “variant” endonuclease, i.e. anendonuclease that does not naturally exist in nature and that isobtained by genetic engineering or by random mutagenesis can bind DNAsequences different from that recognized by wild-type endonucleases (seeinternational application WO2006/097854).

Said rare-cutting endonuclease can be a modular DNA binding nuclease. Bymodular DNA binding nuclease is meant any fusion proteins comprising atleast one catalytic domain of an endonuclease and at least one DNAbinding domain or protein specifying a nucleic acid target sequence. TheDNA binding domain is generally a RNA or DNA-binding domain formed by anindependently folded polypeptide or protein domain that contains atleast one motif that recognizes double- or single-strandedpolynucleotides. Many such polypeptides have been described in the arthaving the ability to bind specific nucleic acid sequences. Such bindingdomains often comprise, as non-limiting examples, helix-turn helixdomains, leucine zipper domains, winged helix domains, helix-loop-helixdomains, HMG-box domains, Immunoglobin domains, B3 domain or engineeredzinc finger domain.

Zinc-Finger Nucleases

Initially developed to cleave DNA in vitro, “Zinc Finger Nucleases”(ZFNs) are a fusion between the cleavage domain of the type IISrestriction enzyme, Fokl, and a DNA recognition domain containing 3 ormore C2H2 zinc finger motifs. The heterodimerization at a particularposition in the DNA of two individual ZFNs in precise orientation andspacing leads to a double-strand break (DSB) in the DNA. The use of suchchimeric endonucleases have been extensively reported in the art asreviewed by Urnov et al. (Genome editing with engineered zinc fingernucleases (2010) Nature reviews Genetics 11:636-646).

Standard ZFNs fuse the cleavage domain to the C-terminus of each zincfinger domain. In order to allow the two cleavage domains to dimerizeand cleave DNA, the two individual ZFNs bind opposite strands of DNAwith their C-termini a certain distance apart. The most commonly usedlinker sequences between the zinc finger domain and the cleavage domainrequires the 5′ edge of each binding site to be separated by 5 to 7 bp.

The most straightforward method to generate new zinc-finger arrays is tocombine smaller zinc-finger “modules” of known specificity. The mostcommon modular assembly process involves combining three separate zincfingers that can each recognize a 3 base pair DNA sequence to generate a3-finger array that can recognize a 9 base pair target site. Numerousselection methods have been used to generate zinc-finger arrays capableof targeting desired sequences. Initial selection efforts utilized phagedisplay to select proteins that bound a given DNA target from a largepool of partially randomized zinc-finger arrays. More recent effortshave utilized yeast one-hybrid systems, bacterial one-hybrid andtwo-hybrid systems, and mammalian cells.

TAL-Nucleases

“TALE-nuclease” or “MBBBD-nuclease” refers to engineered proteinsresulting from the fusion of a DNA binding domain typically derived fromTranscription Activator Like Effector proteins (TALE) or ModularBase-per-Base Binding domain (MBBBD), with a catalytic domain havingendonuclease activity. Such catalytic domain usually comes from enzymes,such as for instance I-Tevl, ColE7, NucA and Fok-I. TALE-nuclease can beformed under monomeric or dimeric forms depending of the selectedcatalytic domain (WO2012138927). Such engineered TALE-nucleases arecommercially available under the trade name TALEN™ (Cellectis, 8 rue dela Croix Jarry, 75013 Paris, France).

According to a preferred embodiment of the invention, the DNA bindingdomain is derived from a Transcription Activator like Effector (TALE),wherein sequence specificity is driven by a series of 33-35 amino acidsrepeats originating from Xanthomonas or Ralstonia bacterial proteinsAvrBs3, PthXo1, AvrHahl, PthA, Tallc as non-limiting examples.

These repeats differ essentially by two amino acids positions thatspecify an interaction with a base pair (Boch, Scholze et al. 2009;Moscou and Bogdanove 2009). Each base pair in the DNA target iscontacted by a single repeat, with the specificity resulting from thetwo variant amino acids of the repeat (the so-called repeat variabledipeptide, RVD). TALE binding domains may further comprise an N-terminaltranslocation domain responsible for the requirement of a first thyminebase (T0) of the targeted sequence and a C-terminal domain thatcontaining a nuclear localization signals (NLS). A TALE nucleic acidbinding domain generally corresponds to an engineered core TALE scaffoldcomprising a plurality of TALE repeat sequences, each repeat comprisinga RVD specific to each nucleotides base of a TALE recognition site. Inthe present invention, each TALE repeat sequence of said core scaffoldis made of 30 to 42 amino acids, more preferably 33 or 34 wherein twocritical amino acids (the so-called repeat variable dipeptide, RVD)located at positions 12 and 13 mediates the recognition of onenucleotide of said TALE binding site sequence; equivalent two criticalamino acids can be located at positions other than 12 and 13 speciallyin TALE repeat sequence taller than 33 or 34 amino acids long.Preferably, RVDs associated with recognition of the differentnucleotides are HD for recognizing C, NG for recognizing T, NI forrecognizing A, NN for recognizing G or A. In another embodiment,critical amino acids 12 and 13 can be mutated towards other amino acidresidues in order to modulate their specificity towards nucleotides A,T, C and G and in particular to enhance this specificity. A TALE nucleicacid binding domain usually comprises between 8 and 30 TALE repeatsequences. More preferably, said core scaffold of the present inventioncomprises between 8 and 20 TALE repeat sequences; again more preferably15 TALE repeat sequences. It can also comprise an additional singletruncated TALE repeat sequence made of 20 amino acids located at theC-terminus of said set of TALE repeat sequences, i.e. an additionalC-terminal half-TALE repeat sequence.

Other engineered DNA binding domains can be used as alternativesequences to form so-called modular base-per-base specific nucleic acidbinding domains (MBBBD) as described in WO 2014/018601. Said MBBBD canbe engineered, for instance, from newly identified proteins, namelyEAV36_BURRH, E5AW43_BURRH, E5AW45_BURRH and E5AW46_BURRH proteins fromthe recently sequenced genome of the endosymbiont fungi BurkholderiaRhizoxinica (Lackner, Moebius et al. 2011). These nucleic acid bindingpolypeptides comprise modules of about 31 to 33 amino acids that arebase specific. These modules display less than 40% sequence identitywith Xanthomonas TALE common repeats and present more polypeptidessequence variability. The different domains from the above proteins(modules, N and C-terminals) from Burkholderia and Xanthomonas areuseful to engineer new proteins or scaffolds having binding propertiesto specific nucleic acid sequences and may be combined to form chimericTALE-MBBBD proteins.

As examples, the present invention encompasses a method for engineeredT-cells in order to inactivate the expression of the genes encodingantigen markers such as CD38, CS1 and CD70 by using specificTALE-nucleases.

Particularly suitable for the realization of the invention,TALE-nucleases such as the ones in SEQ ID NO: 2-3; 5-6; 8-9, SEQ ID NO:64-65; 67-68; 70-71 and SEQ ID NO: 73-74; 76-77; 79-80 for respectivelyCD38, CS1 and CD70 genes. These specific TALE-nucleases, their sequencetarget and the protocol used are presented more thoroughly in thefollowing Examples 1-3.

Delivery Methods

The inventors have considered any means known in the art to allowdelivery inside cells or subcellular compartments of said cells thepolynucleotides expressing the endonucleases, their possibleco-effectors (e.g. guide RNA or DNA associated with Cas9 or Argonautenucleases) as well as the chimeric antigen receptors. These meansinclude viral transduction, electroporation and also liposomal deliverymeans, polymeric carriers, chemical carriers, lipoplexes, polyplexes,dendrimers, nanoparticles, emulsion, natural endocytosis or phagocytosepathway as non-limiting examples.

As a preferred embodiment of the invention, polynucleotides encoding theendonucleases of the present invention are transfected under mRNA formin order to obtain transient expression and avoid chromosomalintegration of foreign DNA, for example by electroporation. Theinventors have determined different optimal conditions for mRNAelectroporation in T-cell displayed in Table 1. The inventor used thecytoPulse technology which allows, by the use of pulsed electric fields,to transiently permeabilize living cells for delivery of material intothe cells (U.S. Pat. No. 6,010,613 and WO 2004/083379). Pulse duration,intensity as well as the interval between pulses can be modified inorder to reach the best conditions for high transfection efficiency withminimal mortality. Basically, the first high electric field pulses allowpore formation, while subsequent lower electric field pulses allow tomoving the polynucleotide into the cell. In one aspect of the presentinvention, the inventor describe the steps that led to achievementof >95% transfection efficiency of mRNA in T cells, and the use of theelectroporation protocol to transiently express different kind ofproteins in T cells. In particular the invention relates to a method oftransforming T cell comprising contacting said T cell with RNA andapplying to T cell an agile pulse sequence consisting of:

-   -   (a) one electrical pulse with a voltage range from 2250 to 3000        V per centimeter, a pulse width of 0.1 ms and a pulse interval        of 0.2 to 10 ms between the electrical pulses of step (a) and        (b);    -   (b) one electrical pulse with a voltage range from 2250 to 3000        V with a pulse width of 100 ms and a pulse interval of 100 ms        between the electrical pulse of step (b) and the first        electrical pulse of step (c); and    -   (c) 4 electrical pulses with a voltage of 325 V with a pulse        width of 0.2 ms and a pulse interval of 2 ms between each of 4        electrical pulses.        In particular embodiment, the method of transforming T cell        comprising contacting said T cell with RNA and applying to T        cell an agile pulse sequence consisting of:    -   (a) one electrical pulse with a voltage of 2250, 2300, 2350,        2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900        or 3000V per centimeter, a pulse width of 0.1 ms and a pulse        interval of 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ms between        the electrical pulses of step (a) and (b);    -   (b) one electrical pulse with a voltage range from 2250, of        2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500,        2600, 2700, 2800, 2900 or 3000V with a pulse width of 100 ms and        a pulse interval of 100 ms between the electrical pulse of        step (b) and the first electrical pulse of step (c); and    -   (c) 4 electrical pulses with a voltage of 325 V with a pulse        width of 0.2 ms and a pulse interval of 2 ms between each of 4        electrical pulses.        Any values included in the value range described above are        disclosed in the present application. Electroporation medium can        be any suitable medium known in the art. Preferably, the        electroporation medium has conductivity in a range spanning 0.01        to 1.0 milliSiemens.

TABLE 1 Different cytopulse programs used to determine the minimalvoltage required for electroporation in PBMC derived T-cells. Cyto-Group 1 Group 2 Group 3 pulse duration Interval duration Intervalduration Interval program Pulses V (ms) (ms) Pulses V (ms) (ms) Pulses V(ms) (ms) 1 1 600 0.1 0.2 1 600 0.1 100 4 130 0.2 2 2 1 900 0.1 0.2 1900 0.1 100 4 130 0.2 2 3 1 1200 0.1 0.2 1 1200 0.1 100 4 130 0.2 2 4 11200 0.1 10 1 900 0.1 100 4 130 0.2 2 5 1 900 0.1 20 1 600 0.1 100 4 1300.2 2

Viral Transduction

According to the present invention, the use of retroviral vectors andmore preferably of lentiviral vectors is particularly suited forexpressing the chimeric antigen receptors into the T-cells. Methods forviral transduction are well known in the art (Walther et al. (2000)Viral Vectors for Gene Transfer. Drugs. 60(2):249-271). IntegrativeViral Vectors Allow the Stable Integration of the polynucleotides in theT-cells genome and to expressing the chimeric antigen receptors over alonger period of time.

Non Alloreactive T Cells

Although the method of the invention could be carried out in-vivo aspart of a gene therapy, for instance, by using viral vectors targetingT-cells in blood circulation, which would include genetic sequencesexpressing a specific rare-cutting endonuclease along with other geneticsequences expressing a CAR, the method of the invention is moregenerally intended to be practiced ex-vivo on cultured T-cellsobtainable from patients or donors. The engineered T-cells engineeredex-vivo can be either re-implanted into a patient from where theyoriginate, as part of an autologous treatment, or to be used as part ofan allogeneic treatment. In this later case, it is preferable to furtherengineer the cells to make them non-alloreactive to ensure their properengraftment. Accordingly, the method of the invention may includeadditional steps of procuring the T-cells from a donor and to inactivategenes thereof involved in MHC recognition and or being targets ofimmunosuppressive drugs such as described for instance in WO2013/176915.

T cell receptors (TCR) are cell surface receptors that participate inthe activation of T cells in response to the presentation of antigen.The TCR is generally made from two chains, alpha and beta, whichassemble to form a heterodimer and associates with the CD3-transducingsubunits to form the T-cell receptor complex present on the cellsurface. Each alpha and beta chain of the TCR consists of animmunoglobulin-like N-terminal variable (V) and constant (C) region, ahydrophobic transmembrane domain, and a short cytoplasmic region. As forimmunoglobulin molecules, the variable region of the alpha and betachains are generated by V(D)J recombination, creating a large diversityof antigen specificities within the population of T cells. However, incontrast to immunoglobulins that recognize intact antigen, T cells areactivated by processed peptide fragments in association with an MHCmolecule, introducing an extra dimension to antigen recognition by Tcells, known as MHC restriction. Recognition of MHC disparities betweenthe donor and recipient through the T cell receptor leads to T cellproliferation and the potential development of GVHD. It has been shownthat normal surface expression of the TCR depends on the coordinatedsynthesis and assembly of all seven components of the complex (Ashwelland Klusner 1990). The inactivation of TCRalpha or TCRbeta can result inthe elimination of the TCR from the surface of T cells preventingrecognition of alloantigen and thus GVHD.

Thus, still according to the invention, engraftment of the T-cells maybe improved by inactivating at least one gene encoding a TCR component.TCR is rendered not functional in the cells by inactivating TCR alphagene and/or TCR beta gene(s).

With respect to the use of Cas9/CRISPR system, the inventors havedetermined appropriate target sequences within the 3 exons encoding TCR,allowing a significant reduction of toxicity in living cells, whileretaining cleavage efficiency. The preferred target sequences are notedin Table 2 (+ for lower ratio of TCR negative cells, ++ for intermediateratio, +++ for higher ratio).

TABLE 2appropriate target sequences for the guide RNA using Cas9 in T-cellsExon TCR Position Strand Target genomic sequence SEQ ID efficiency Ex178 −1 GAGAATCAAAATCGGTGAATAGG 102 +++ Ex3 26 1 TTCAAAACCTGTCAGTGATTGGG103 +++ Ex1 153 1 TGTGCTAGACATGAGGTCTATGG 104 +++ Ex3 74 −1CGTCATGAGCAGATTAAACCCGG 105 +++ Ex1 4 −1 TCAGGGTTCTGGATATCTGTGGG 106 +++Ex1 5 −1 GTCAGGGTTCTGGATATCTGTGG 107 +++ Ex3 33 −1TTCGGAACCCAATCACTGACAGG 108 +++ Ex3 60 −1 TAAACCCGGCCACTTTCAGGAGG 109+++ Ex1 200 −1 AAAGTCAGATTTGTTGCTCCAGG 110 ++ Ex1 102 1AACAAATGTGTCACAAAGTAAGG 111 ++ Ex1 39 −1 TGGATTTAGAGTCTCTCAGCTGG 112 ++Ex1 59 −1 TAGGCAGACAGACTTGTCACTGG 113 ++ Ex1 22 −1AGCTGGTACACGGCAGGGTCAGG 114 ++ Ex1 21 −1 GCTGGTACACGGCAGGGTCAGGG 115 ++Ex1 28 −1 TCTCTCAGCTGGTACACGGCAGG 116 ++ Ex3 25 1TTTCAAAACCTGTCAGTGATTGG 117 ++ Ex3 63 −1 GATTAAACCCGGCCACTTTCAGG 118 ++Ex2 17 −1 CTCGACCAGCTTGACATCACAGG 119 ++ Ex1 32 −1AGAGTCTCTCAGCTGGTACACGG 120 ++ Ex1 27 −1 CTCTCAGCTGGTACACGGCAGGG 121 ++Ex2 12 1 AAGTTCCTGTGATGTCAAGCTGG 122 ++ Ex3 55 1 ATCCTCCTCCTGAAAGTGGCCGG123 ++ Ex3 86 1 TGCTCATGACGCTGCGGCTGTGG 124 ++ Ex1 146 1ACAAAACTGTGCTAGACATGAGG 125 + Ex1 86 −1 ATTTGTTTGAGAATCAAAATCGG 126 +Ex2 3 −1 CATCACAGGAACTTTCTAAAAGG 127 + Ex2 34 1 GTCGAGAAAAGCTTTGAAACAGG128 + Ex3 51 −1 CCACTTTCAGGAGGAGGATTCGG 129 + Ex3 18 −1CTGACAGGTTTTGAAAGTTTAGG 130 + Ex2 43 1 AGCTTTGAAACAGGTAAGACAGG 131 + Ex1236 −1 TGGAATAATGCTGTTGTTGAAGG 132 + Ex1 182 1 AGAGCAACAGTGCTGTGGCCTGG133 + Ex3 103 1 CTGTGGTCCAGCTGAGGTGAGGG 134 + Ex3 97 1CTGCGGCTGTGGTCCAGCTGAGG 135 + Ex3 104 1 TGTGGTCCAGCTGAGGTGAGGGG 136 +Ex1 267 1 CTTCTTCCCCAGCCCAGGTAAGG 137 + Ex1 15 −1ACACGGCAGGGTCAGGGTTCTGG 138 + Ex1 177 1 CTTCAAGAGCAACAGTGCTGTGG 139 +Ex1 256 −1 CTGGGGAAGAAGGTGTCTTCTGG 140 + Ex3 56 1TCCTCCTCCTGAAAGTGGCCGGG 141 + Ex3 80 1 TTAATCTGCTCATGACGCTGCGG 142 + Ex357 −1 ACCCGGCCACTTTCAGGAGGAGG 143 + Ex1 268 1 TTCTTCCCCAGCCCAGGTAAGGG144 + Ex1 266 −1 CTTACCTGGGCTGGGGAAGAAGG 145 + Ex1 262 1GACACCTTCTTCCCCAGCCCAGG 146 + Ex3 102 1 GCTGTGGTCCAGCTGAGGTGAGG 147 +Ex3 51 1 CCGAATCCTCCTCCTGAAAGTGG 148 +

MHC antigens are also proteins that played a major role intransplantation reactions. Rejection is mediated by T cells reacting tothe histocompatibility antigens on the surface of implanted tissues, andthe largest group of these antigens is the major histocompatibilityantigens (MHC). These proteins are expressed on the surface of allhigher vertebrates and are called HLA antigens (for human leukocyteantigens) in human cells. Like TCR, the MHC proteins serve a vital rolein T cell stimulation. Antigen presenting cells (often dendritic cells)display peptides that are the degradation products of foreign proteinson the cell surface on the MHC. In the presence of a co-stimulatorysignal, the T cell becomes activated, and will act on a target cell thatalso displays that same peptide/MHC complex. For example, a stimulated Thelper cell will target a macrophage displaying an antigen inconjunction with its MHC, or a cytotoxic T cell (CTL) will act on avirally infected cell displaying foreign viral peptides.

Thus, in order to provide less alloreactive T-cells, the method of theinvention can further comprise the step of inactivating or mutating oneHLA gene.

The class I HLA gene cluster in humans comprises three major loci, B, Cand A, as well as several minor loci. The class II HLA cluster alsocomprises three major loci, DP, DQ and DR, and both the class I andclass II gene clusters are polymorphic, in that there are severaldifferent alleles of both the class I and II genes within thepopulation. There are also several accessory proteins that play a rolein HLA functioning as well. The Tapl and Tap2 subunits are parts of theTAP transporter complex that is essential in loading peptide antigens onto the class I HLA complexes, and the LMP2 and LMP7 proteosome subunitsplay roles in the proteolytic degradation of antigens into peptides fordisplay on the HLA. Reduction in LMP7 has been shown to reduce theamount of MHC class I at the cell surface, perhaps through a lack ofstabilization (Fehling et al. (1999) Science 265:1234-1237). In additionto TAP and LMP, there is the tapasin gene, whose product forms a bridgebetween the TAP complex and the HLA class I chains and enhances peptideloading. Reduction in tapasin results in cells with impaired MHC class Iassembly, reduced cell surface expression of the MHC class I andimpaired immune responses (Grandea et al. (2000) Immunity 13:213-222 andGarbi et al. (2000) Nat. Immunol. 1:234-238). Any of the above genes maybe inactivated as part of the present invention as disclosed, forinstance in WO 2012/012667.

Method of Engineering Drug-Resistant T-Cells:

To improve cancer therapy and selective engraftment of allogeneicT-cells, drug resistance can be conferred to the engineered T-cells toprotect them from the toxic side effects of chemotherapy orimmunosuppressive agents. Indeed, the inventors have observed that mostpatients were treated with chemotherapy and immune depleting agents as astandard of care, prior to receiving T-cell immunotherapy. Also theyfound that they could take advantage of these treatments to help theselection of the engineered T-cells, either by adding chemotherapy drugsin culture media for expansion of the cells ex-vivo prior to treatment,or by obtaining a selective expansion of the engineered T-cells in-vivoin patients under chemotherapy or immunosuppressive treatments.

Also the drug resistance of T-cells also permits their enrichment in orex vivo, as T-cells which express the drug resistance gene, will surviveand multiply relative to drug sensitive cells. In particular, thepresent invention relates to a method of engineering allogeneic and drugresistance T-cells resistant for immunotherapy comprising:

(a) Providing a T-cell;

(b) Selecting at least one drug;

(c) Modifying T-cell to confer drug resistance to said T-cell;

(d) Expanding said engineered T-cell in the presence of said drug, andoptionally the preceding steps may be combined with the steps of themethods as previously described.

Drug resistance can be conferred to a T-cell by inactivating one or moregene(s) responsible for the cell's sensitivity to the drug (drugsensitizing gene(s)), such as the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (Genbank: M26434.1). In particular HPRT can beinactivated in engineered T-cells to confer resistance to a cytostaticmetabolite, the 6-thioguanine (6TG) which is converted by HPRT tocytotoxic thioguanine nucleotide and which is currently used to treatpatients with cancer, in particular leukemias (Hacke, Treger et al.2013). Another example if the inactivation of the CD3 normally expressedat the surface of the T-cell can confer resistance to anti-CD3antibodies such as teplizumab.

Drug resistance can also be conferred to a T-cell by expressing a drugresistance gene. Said drug resistance gene refers to a nucleic acidsequence that encodes “resistance” to an agent, such as achemotherapeutic agent (e.g. methotrexate). In other words, theexpression of the drug resistance gene in a cell permits proliferationof the cells in the presence of the agent to a greater extent than theproliferation of a corresponding cell without the drug resistance gene.A drug resistance gene of the invention can encode resistance toanti-metabolite, methotrexate, vinblastine, cisplatin, alkylatingagents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, theiranalogs or derivatives, and the like.

Variant alleles of several genes such as dihydrofolate reductase (DHFR),inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin ormethylguanine transferase (MGMT) have been identified to confer drugresistance to a cell. Said drug resistance gene can be expressed in thecell either by introducing a transgene encoding said gene into the cellor by integrating said drug resistance gene into the genome of the cellby homologous recombination. Several other drug resistance genes havebeen identified that can potentially be used to confer drug resistanceto targeted cells (Takebe, Zhao et al. 2001; Sugimoto, Tsukahara et al.2003; Zielske, Reese et al. 2003; Nivens, Felder et al. 2004;Bardenheuer, Lehmberg et al. 2005; Kushman, Kabler et al. 2007).

DHFR is an enzyme involved in regulating the amount of tetrahydrofolatein the cell and is essential to DNA synthesis. Folate analogs such asmethotrexate (MTX) inhibit DHFR and are thus used as anti-neoplasticagents in clinic. Different mutant forms of DHFR which have increasedresistance to inhibition by anti-folates used in therapy have beendescribed. In a particular embodiment, the drug resistance geneaccording to the present invention can be a nucleic acid sequenceencoding a mutant form of human wild type DHFR (GenBank: AAH71996.1)which comprises at least one mutation conferring resistance to ananti-folate treatment, such as methotrexate. In particular embodiment,mutant form of DHFR comprises at least one mutated amino acid atposition G15, L22, F31 or F34, preferably at positions L22 or F31((Schweitzer, Dicker et al. 1990); International application WO94/24277; U.S. Pat. No. 6,642,043).

As used herein, “antifolate agent” or “folate analogs” refers to amolecule directed to interfere with the folate metabolic pathway at somelevel. Examples of antifolate agents include, e.g., methotrexate (MTX);aminopterin; trimetrexate (Neutrexin™); edatrexate;N10-propargyl-5,8-dideazafolic acid (CB3717); ZD1694 (Tumodex),5,8-dideazaisofolic acid (IAHQ); 5,10-dideazatetrahydrofolic acid(DDATHF); 5-deazafolic acid; PT523 (N alpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloyl-L-ornithine); 10-ethyl-10-deazaaminopterin (DDATHF,lomatrexol); piritrexim; 10-EDAM; ZD1694; GW1843; Pemetrexate and PDX(10-propargyl-10-deazaaminopterin).

Another example of drug resistance gene can also be a mutant or modifiedform of ionisine-5′-monophosphate dehydrogenase II (IMPDH2), arate-limiting enzyme in the de novo synthesis of guanosine nucleotides.The mutant or modified form of IMPDH2 is a IMPDH inhibitor resistancegene. IMPDH inhibitors can be mycophenolic acid (MPA) or its prodrugmycophenolate mofetil (MMF). The mutant IMPDH2 can comprises at leastone, preferably two mutations in the MAP binding site of the wild typehuman IMPDH2 (NP_000875.2) that lead to a significantly increasedresistance to IMPDH inhibitor. The mutations are preferably at positionsT333 and/or S351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al.2007; Jonnalagadda, Brown et al. 2013). In a particular embodiment, thethreonine residue at position 333 is replaced with an isoleucine residueand the serine residue at position 351 is replaced with a tyrosineresidue.

Another drug resistance gene is the mutant form of calcineurin.Calcineurin (PP2B) is an ubiquitously expressed serine/threonine proteinphosphatase that is involved in many biological processes and which iscentral to T-cell activation. Calcineurin is a heterodimer composed of acatalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB;two isoforms). After engagement of the T-cell receptor, calcineurindephosphorylates the transcription factor NFAT, allowing it totranslocate to the nucleus and active key target gene such as IL2. FK506in complex with FKBP12, or cyclosporine A (CsA) in complex with CyPAblock NFAT access to calcineurin's active site, preventing itsdephosphorylation and thereby inhibiting T-cell activation (Brewin,Mancao et al. 2009). The drug resistance gene of the present inventioncan be a nucleic acid sequence encoding a mutant form of calcineurinresistant to calcineurin inhibitor such as FK506 and/or CsA. In aparticular embodiment, said mutant form can comprise at least onemutated amino acid of the wild type calcineurin heterodimer a atpositions: V314, Y341, M347, T351, W352, L354, K360, preferably doublemutations at positions T351 and L354 or V314 and Y341. Correspondence ofamino acid positions described herein is frequently expressed in termsof the positions of the amino acids of the form of wild-type humancalcineurin heterodimer (GenBank: ACX34092.1).

In another particular embodiment, said mutant form can comprise at leastone mutated amino acid of the wild type calcineurin heterodimer b atpositions: V120, N123, L124 or K125, preferably double mutations atpositions L124 and K125. Correspondence of amino acid positionsdescribed herein is frequently expressed in terms of the positions ofthe amino acids of the form of wild-type human calcineurin heterodimer bpolypeptide (GenBank: ACX34095.1).

Another drug resistance gene is 0(6)-methylguanine methyltransferase(MGMT) encoding human alkyl guanine transferase (hAGT). AGT is a DNArepair protein that confers resistance to the cytotoxic effects ofalkylating agents, such as nitrosoureas and temozolomide (TMZ).6-benzylguanine (6-BG) is an inhibitor of AGT that potentiatesnitrosourea toxicity and is co-administered with TMZ to potentiate thecytotoxic effects of this agent. Several mutant forms of MGMT thatencode variants of AGT are highly resistant to inactivation by 6-BG, butretain their ability to repair DNA damage (Maze, Kurpad et al. 1999). Ina particular embodiment, AGT mutant form can comprise a mutated aminoacid of the wild type AGT position P140 (UniProtKB: P16455).

Another drug resistance gene can be multidrug resistance protein 1(MDR1) gene. This gene encodes a membrane glycoprotein, known asP-glycoprotein (P-GP) involved in the transport of metabolic byproductsacross the cell membrane. The P-Gp protein displays broad specificitytowards several structurally unrelated chemotherapy agents. Thus, drugresistance can be conferred to cells by the expression of nucleic acidsequence that encodes MDR-1 (NP_000918).

Drug resistance gene can also be cytotoxic antibiotics, such as ble geneor mcrA gene. Ectopic expression of ble gene or mcrA in an immune cellgives a selective advantage when exposed to the chemotherapeutic agent,respectively the bleomycine or the mitomycin C.

The T-cells can also be made resistant to immunosuppressive agents. Animmunosuppressive agent is an agent that suppresses immune function byone of several mechanisms of action. In other words, animmunosuppressive agent is a role played by a compound which isexhibited by a capability to diminish the extent and/or voracity of animmune response. As non-limiting example, an immunosuppressive agent canbe a calcineurin inhibitor, a target of rapamycin, an interleukin-2α-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, aninhibitor of dihydrofolic acid reductase, a corticosteroid or animmunosuppressive antimetabolite. Classical cytotoxic immunosuppressantsact by inhibiting DNA synthesis. Others may act through activation ofT-cells or by inhibiting the activation of helper cells. The methodaccording to the invention allows conferring immunosuppressiveresistance to T cells for immunotherapy by inactivating the target ofthe immunosuppressive agent in T cells. As non-limiting examples,targets for immunosuppressive agent can be a receptor for animmunosuppressive agent such as: CD52, glucocorticoid receptor (GR), aFKBP family gene member and a cyclophilin family gene member.

In immunocompetent hosts, allogeneic cells are normally rapidly rejectedby the host immune system. It has been demonstrated that, allogeneicleukocytes present in non-irradiated blood products will persist for nomore than 5 to 6 days. Thus, to prevent rejection of allogeneic cells,the host's immune system must be effectively suppressed.Glucocorticoidsteroids are widely used therapeutically forimmunosuppression. This class of steroid hormones binds to theglucocorticoid receptor (GR) present in the cytosol of T cells resultingin the translocation into the nucleus and the binding of specific DNAmotifs that regulate the expression of a number of genes involved in theimmunologic process. Treatment of T cells with glucocorticoid steroidsresults in reduced levels of cytokine production leading to T cellanergy and interfering in T cell activation. Alemtuzumab, also known asCAMPATH1-H, is a humanized monoclonal antibody targeting CD52, a 12amino acid glycosylphosphatidyl-inositol-(GPI) linked glycoprotein(Waldmann and Hale, 2005). CD52 is expressed at high levels on T and Blymphocytes and lower levels on monocytes while being absent ongranulocytes and bone marrow precursors. Treatment with Alemtuzumab, ahumanized monoclonal antibody directed against CD52, has been shown toinduce a rapid depletion of circulating lymphocytes and monocytes. It isfrequently used in the treatment of T cell lymphomas and in certaincases as part of a conditioning regimen for transplantation. However, inthe case of adoptive immunotherapy the use of immunosuppressive drugswill also have a detrimental effect on the introduced therapeutic Tcells. Therefore, to effectively use an adoptive immunotherapy approachin these conditions, the introduced cells would need to be resistant tothe immunosuppressive treatment.

As a preferred embodiment of the above steps, said gene of step (b),specific for an immunosuppressive treatment, is CD52, and theimmunosuppressive treatment of step (d) comprises a humanized antibodytargeting CD52 antigen. As another embodiment, said gene of step (b),specific for an immunosuppressive treatment, is a glucocorticoidreceptor (GR) and the immunosuppressive treatment of step d) comprises acorticosteroid such as dexamethasone. As another embodiment, said targetgene of step (b), specific for an immunosuppressive treatment, is a FKBPfamily gene member or a variant thereof and the immunosuppressivetreatment of step (d) comprises FK506 also known as Tacrolimus orfujimycin. As another embodiment, said FKBP family gene member is FKBP12or a variant thereof. As another embodiment, said gene of step (b),specific for an immunosuppressive treatment, is a cyclophilin familygene member or a variant thereof and the immunosuppressive treatment ofstep (d) comprises cyclosporine.

In a particular embodiment of the invention, the genetic modificationstep of the method relies on the inactivation of two genes selected fromthe group consisting of CD52 and GR, CD52 and TCR alpha, CDR52 and TCRbeta, GR and TCR alpha, GR and TCR beta, TCR alpha and TCR beta. Inanother embodiment, the genetic modification step of the method relieson the inactivation of more than two genes. The genetic modification ispreferably operated ex-vivo using at least two RNA guides targeting thedifferent genes.

By inactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form.

Engineering Highly Active T Cells for Immunotherapy

According to the present invention, the T-cells can be selected from thegroup consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes,regulatory T-lymphocytes or helper T-lymphocytes. In another embodiment,said cell can be derived from the group consisting of CD4+T-lymphocytesand CD8+T-lymphocytes. They can be extracted from blood or derived fromstem cells. The stem cells can be adult stem cells, embryonic stemcells, more particularly non-human stem cells, cord blood stem cells,progenitor cells, bone marrow stem cells, induced pluripotent stemcells, totipotent stem cells or hematopoietic stem cells. Representativehuman cells are CD34+ cells. Prior to expansion and genetic modificationof the cells of the invention, a source of cells can be obtained from asubject through a variety of non-limiting methods. T-cells can beobtained from a number of non-limiting sources, including peripheralblood mononuclear cells, bone marrow, lymph node tissue, cord blood,thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In certain embodiments of thepresent invention, any number of T cell lines available and known tothose skilled in the art, may be used. In another embodiment, said cellcan be derived from a healthy donor, from a patient diagnosed withcancer or from a patient diagnosed with an infection. In anotherembodiment, said cell is part of a mixed population of cells whichpresent different phenotypic characteristics. In the scope of thepresent invention is also encompassed a cell line obtained from atransformed T-cell according to the method previously described.

As a further aspect of the invention, the T-cells according to theinvention may be further engineered, preferably genetically engineered,to enhance their activity and/or activation, especially by modulatingthe expression of proteins involved in overall T-cell regulation,referred to as “immune-checkpoints”.

Immune Check Points

It will be understood by those of ordinary skill in the art, that theterm “immune checkpoints” means a group of molecules expressed by Tcells. These molecules effectively serve as “brakes” to down-modulate orinhibit an immune response. Immune checkpoint molecules include, but arenot limited to Programmed Death 1 (PD-1, also known as PDCD1 or CD279,accession number: NM_005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4,also known as CD152, GenBank accession number AF414120.1), LAG3 (alsoknown as CD223, accession number: NM_002286.5), Tim3 (also known asHAVCR2, GenBank accession number: JX049979.1), BTLA (also known asCD272, accession number: NM_181780.3), BY55 (also known as CD160,GenBank accession number: CR541888.1), TIGIT (also known as IVSTM3,accession number: NM_173799), LAIR1 (also known as CD305, GenBankaccession number: CR542051.1, {Meyaard, 1997 #122}), SIGLEC10 (GeneBankaccession number: AY358337.1), 2B4 (also known as CD244, accessionnumber: NM_001166664.1), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM,SIGLEC7 {Nicoll, 1999 #123}, SIGLEC9 {Zhang, 2000 #124; Ikehara, 2004#125}, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD,FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1,IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3,PRDM1, BATF {Quigley, 2010 #121}, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3which directly inhibit immune cells. For example, CTLA-4 is acell-surface protein expressed on certain CD4 and CD8 T cells; whenengaged by its ligands (B7-1 and B7-2) on antigen presenting cells,T-cell activation and effector function are inhibited. Thus the presentinvention relates to a method of engineering T-cells, especially forimmunotherapy, comprising genetically modifying T-cells by inactivatingat least one protein involved in the immune check-point, in particularPD1 and/or CTLA-4 or any immune-checkpoint proteins referred to in Table3.

TABLE 3 List of genes encoding immune checkpoint proteins. Genes thatcan be inactivated Pathway In the pathway Co-inhibitory CTLA4 (CD152)CTLA4, PPP2CA, PPP2CB, PTPN6, receptors PTPN22 PDCD1 (PD-1, CD279) PDCD1CD223 (lag3) LAG3 HAVCR2 (tim3) HAVCR2 BTLA(cd272) BTLA CD160(by55)CD160 IgSF family TIGIT CD96 CRTAM LAIR1(cd305) LAIR1 SIGLECs SIGLEC7SIGLEC9 CD244(2b4) CD244 Death receptors TRAIL TNFRSF10B, TNFRSF10A,CASP8, CASP10, CASP3, CASP6, CASP7 FAS FADD, FAS Cytokine signallingTGF-beta signaling TGFBRII, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI,SKIL, TGIF1 IL10 signalling IL10RA, IL10RB, HMOX2 IL6 signalling IL6R,IL6ST Arginine/tryptophan EIF2AK4 starvation Prevention of TCR CSK, PAG1signalling SIT1 Induced Treg induced Treg FOXP3 Transcriptiontranscription factors PRDM1 (=blimp1, heterozygotes mice factorscontrolling exhaustion control chronic viral infection bettercontrolling than wt or conditional KO) exhaustion BATF Hypoxia mediatediNOS induced guanylated GUCY1A2, GUCY1A3, GUCY1B2, tolerance cyclaseGUCY1B3

Engineered T-Cells Expressing Chimeric Antigen Receptors AgainstPathological Cells

The chimeric antigen receptors introduced into the T-cells according tothe invention can adopt different design such as single-chain ormulti-chain CARs. These different designs allow various strategies forimproving specificity and binding efficiency towards the targetedpathological cells. Some of these strategies are illustrated in thefigures of the present application. Single-chain CARs are the mostclassical version in the art. Multi-chain CAR architectures weredeveloped by the applicant as allowing modulation of the activity ofT-cells in terms of specificity and intensity. The multiple subunits canshelter additional co-stimulation domains or keep such domains at adistance, as well as other types of receptors, whereas classical singlechain architecture can sometimes be regarded as too much sensitive andless permissive to multispecific interactions.

Single-Chain CAR

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T cells generated ex vivo, is a promising strategy totreat viral infections and cancer. The T cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specific Tcells or redirection of T cells through genetic engineering (Park,Rosenberg et al. 2011). Transfer of viral antigen specific T cells is awell-established procedure used for the treatment of transplantassociated viral infections and rare viral-related malignancies.Similarly, isolation and transfer of tumor specific T cells has beenshown to be successful in treating melanoma.

Novel specificities in T cells have been successfully generated throughthe genetic transfer of transgenic T cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity. However, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T cells.CARs have successfully allowed T cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

In addition to the CAR targeting the antigen marker, which is common tothe pathological cells and the T-cells, such as CD38, it is envisionedto express further CARs directed towards other antigen markers notnecessarily expressed by the T-cells, so as to enhancing T-cellsspecificity.

Examples of chimeric antigen receptor that can be further expressed bythe T-cells to create multi-specific cells, are antigen receptorsdirected against multiple myeloma or lymphoblastic leukemia antigenmarkers, such as TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25),GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROTP53708), and FCRL5 (UNIPROT Q685N8).

As further examples, the antigen of the target can be from any clusterof differentiation molecules (e.g. CD16, CD64, CD78, CD96, CLL1, CD116,CD117, CD71, CD45, CD71, CD123 and CD138), a tumor-associated surfaceantigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA),epithelial cell adhesion molecule (EpCAM), epidermal growth factorreceptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40,disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72,glycosphingolipids, glioma-associated antigen, β-human chorionicgonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF,prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53,prostein, PSMA, surviving and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22,insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, amajor histocompatibility complex (MHC) molecule presenting atumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromalantigens, the extra domain A (EDA) and extra domain B (EDB) offibronectin and the A1 domain of tenascin-C (TnC A1) and fibroblastassociated protein (fap); a lineage-specific or tissue specific antigensuch as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4,B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a majorhistocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), or avirus-specific surface antigen such as an HIV-specific antigen (such asHIV gp120); an EBV-specific antigen, a CMV-specific antigen, aHPV-specific antigen, a Lasse Virus-specific antigen, an InfluenzaVirus-specific antigen as well as any derivate or variant of thesesurface markers. Antigens are not necessarily surface marker antigensbut can be also endogenous small antigens presented by HLA class I atthe surface of the cells.

As examples, the present invention encompasses single-chain CARs whichtarget specifically cell surface marker, such as CD38, CS1 and/or CD70as described in the examples, together with an inactivation of the genesencoding respectively CD38, CS1 and/or CD70 in the cells expressing saidCARs.

As a specific example, the VH and VL chains of the scFv anti-CD38 shareat least 80%, preferably 90% and more preferably 95% of identity withrespectively SEQ ID NO:10 and 12 and SEQ ID NO: 11 and 13.

As a specific example, the antibody or epitope-binding on CD38 antigen,characterized in that said antibody or epitope-binding fragment thereofcomprises at least one heavy chain and at least one light chain, whereinsaid heavy chain comprises three sequential complementarity-determiningregions having amino acid sequences represented by SEQ ID NOS: 14-17,and wherein said light chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOS: 21-23.

As a another specific example, the antibody or epitope-binding on CD38antigen, characterized in that said antibody or epitope-binding fragmentthereof comprises at least one heavy chain and at least one light chain,wherein said heavy chain comprises three sequentialcomplementarity-determining regions having amino acid sequencesrepresented by SEQ ID NOS: 18-20, and wherein said light chain comprisesthree sequential complementarity-determining regions having amino acidsequences represented by SEQ ID NOS: 24-26.

As another specific example, the VH and VL chains of the scFv anti-CS1share at least 80%, preferably 90% and more preferably 95% of identitywith respectively SEQ ID NO:38-40-42-44-46 and SEQ ID NO:39-41-42-45-46.

As still another specific example, the VH and VL chains of the scFvanti-CD70 share at least 80%, preferably 90% and more preferably 95% ofidentity at the polynucleotide or nucleic acid level with respectivelySEQ ID NO:81-82; 85-86; 89-91 and SEQ ID NO: 83-84; 87-88; 91-92.

In an embodiment, the invention encompasses a polynucleotide encoding asingle CAR anti-CD38 which shares at least 80%, preferably 90% and morepreferably 95% of identity with SEQ ID NO: 35-37. In another embodiment,the invention encompassed a polynucleotide encoding a single CARanti-CS1 which shares at least 80%, preferably 90% and more preferably95% of identity with SEQ ID NO: 48-62.

In still another embodiment, the invention encompasses a polynucleotideencoding a single CAR anti-CD70 which shares at least 80%, preferably90% and more preferably 95% of identity with SEQ ID NO: 93-101.

The present invention is more particularly drawn to immune cells thatare endowed with a CAR presenting some identity with those described inthe present application and that would bear rare-cutting endonucleaseinduced mutations in a gene encoding the cell marker targeted by saidCAR (i.e. the CAR displays affinity with the product of said inactivatedgene). By identity is meant at least 70%, preferably 80%, morepreferably 90% and even more preferably 95% polynucleotide orpolypeptide identity as determined by the software such as FASTA, orBLAST which are available as a part of the GCG sequence analysis package(University of Wisconsin, Madison, Wis.). BLASTP “Identities” shows thenumber and fraction of total residues in the high scoring sequence pairswhich are identical. Amino acid sequences having these degrees ofidentity or similarity or any intermediate degree of identity ofsimilarity to the amino acid sequences disclosed herein are contemplatedand encompassed by this disclosure. The same applies with respect topolynucleotide sequences using BLASTN.

Multi-Subunit CAR

Chimeric antigen receptors from the prior art introduced in T-cells havebeen formed of single chain polypeptides that necessitate serialappending of signaling domains. However, by moving signaling domainsfrom their natural juxtamembrane position may interfere with theirfunction. To overcome this drawback, the applicant recently designed amulti-chain CAR derived from FcERI to allow normal juxtamembraneposition of all relevant signaling domains. In this new architecture,the high affinity IgE binding domain of FcERI alpha chain is replaced byan extracellular ligand-binding domain such as scFv to redirect T-cellspecificity against cell targets and the N and/or C-termini tails ofFcERI beta chain are used to place costimulatory signals in normaljuxtamembrane positions.

Accordingly, the CAR expressed by the engineered T-cell according to theinvention can be a multi-chain chimeric antigen receptor (CAR)particularly adapted to the production and expansion of engineeredT-cells of the present invention. Such multi-chain CARs comprise atleast two of the following components:

-   -   a) one polypeptide comprising the transmembrembrane domain of        FcERI alpha chain and an extracellular ligand-binding domain,    -   b) one polypeptide comprising a part of N- and C-terminal        cytoplasmic tail and the transmembrane domain of FcERI beta        chain and/or    -   c) at least two polypeptides comprising each a part of        intracytoplasmic tail and the transmembrane domain of FcERI        gamma chain, whereby different polypeptides multimerize together        spontaneously to form dimeric, trimeric or tetrameric CAR.

According to such architectures, ligands binding domains and signalingdomains are born on separate polypeptides. The different polypeptidesare anchored into the membrane in a close proximity allowinginteractions with each other. In such architectures, the signaling andco-stimulatory domains can be in juxtamembrane positions (i.e. adjacentto the cell membrane on the internal side of it), which is deemed toallow improved function of co-stimulatory domains. The multi-subunitarchitecture also offers more flexibility and possibilities of designingCARs with more control on T-cell activation. For instance, it ispossible to include several extracellular antigen recognition domainshaving different specificity to obtain a multi-specific CARarchitecture. It is also possible to control the relative ratio betweenthe different subunits into the multi-chain CAR. This type ofarchitecture has been recently described by the applicant inPCT/US2013/058005 (WO2014/039523).

The assembly of the different chains as part of a single multi-chain CARis made possible, for instance, by using the different alpha, beta andgamma chains of the high affinity receptor for IgE (FcERI) (Metzger,Alcaraz et al. 1986) to which are fused the signaling and co-stimulatorydomains. The gamma chain comprises a transmembrane region andcytoplasmic tail containing one immunoreceptor tyrosine-based activationmotif (ITAM) (Cambier 1995).

The multi-chain CAR can comprise several extracellular ligand-bindingdomains, to simultaneously bind different elements in target therebyaugmenting immune cell activation and function. In one embodiment, theextracellular ligand-binding domains can be placed in tandem on the sametransmembrane polypeptide, and optionally can be separated by a linker.In another embodiment, said different extracellular ligand-bindingdomains can be placed on different transmembrane polypeptides composingthe multi-chain CAR. In another embodiment, the present inventionrelates to a population of multi-chain CARs comprising each onedifferent extracellular ligand binding domains. In a particular, thepresent invention relates to a method of engineering immune cellscomprising providing an immune cell and expressing at the surface ofsaid cell a population of multi-chain CAR each one comprising differentextracellular ligand binding domains. In another particular embodiment,the present invention relates to a method of engineering an immune cellcomprising providing an immune cell and introducing into said cellpolynucleotides encoding polypeptides composing a population ofmulti-chain CAR each one comprising different extracellular ligandbinding domains. In a particular embodiment the method of engineering animmune cell comprises expressing at the surface of the cell at least apart of FcERI beta and/or gamma chain fused to a signal-transducingdomain and several part of FcERI alpha chains fused to differentextracellular ligand binding domains. In a more particular embodiment,said method comprises introducing into said cell at least onepolynucleotide which encodes a part of FcERI beta and/or gamma chainfused to a signal-transducing domain and several FcERI alpha chainsfused to different extracellular ligand binding domains. By populationof multi-chain CARs, it is meant at least two, three, four, five, six ormore multi-chain CARs each one comprising different extracellular ligandbinding domains. The different extracellular ligand binding domainsaccording to the present invention can preferably simultaneously binddifferent elements in target thereby augmenting immune cell activationand function.

The present invention also relates to an isolated immune cell whichcomprises a population of multi-chain CARs each one comprising differentextracellular ligand binding domains.

The signal transducing domain or intracellular signaling domain of themulti-chain CAR of the invention is responsible for intracellularsignaling following the binding of extracellular ligand binding domainto the target resulting in the activation of the immune cell and immuneresponse. In other words, the signal transducing domain is responsiblefor the activation of at least one of the normal effector functions ofthe immune cell in which the multi-chain CAR is expressed. For example,the effector function of a T cell can be a cytolytic activity or helperactivity including the secretion of cytokines.

In the present application, the term “signal transducing domain” refersto the portion of a protein which transduces the effector signalfunction signal and directs the cell to perform a specialized function.

Preferred examples of signal transducing domain for use in single ormulti-chain CAR can be the cytoplasmic sequences of the Fc receptor or Tcell receptor and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivate or variant of these sequences and any synthetic sequence thatas the same functional capability. Signal transduction domain comprisestwo distinct classes of cytoplasmic signaling sequence, those thatinitiate antigen-dependent primary activation, and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal. Primary cytoplasmic signaling sequence can comprise signalingmotifs which are known as immunoreceptor tyrosine-based activationmotifs of ITAMs. ITAMs are well defined signaling motifs found in theintracytoplasmic tail of a variety of receptors that serve as bindingsites for syk/zap70 class tyrosine kinases. Examples of ITAM used in theinvention can include as non-limiting examples those derived fromTCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon,CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, thesignaling transducing domain of the multi-chain CAR can comprise theCD3zeta signaling domain, or the intracytoplasmic domain of the FcERIbeta or gamma chains.

In particular embodiment the signal transduction domain of themulti-chain CAR of the present invention comprises a co-stimulatorysignal molecule. A co-stimulatory molecule is a cell surface moleculeother than an antigen receptor or their ligands that is required for anefficient immune response.

Ligand binding-domains can be any antigen receptor previously used, andreferred to, with respect to single-chain CAR referred to in theliterature, in particular scFv from monoclonal antibodies. Bispecific ormulti-specific CARs as described in WO 2014/4011988 are incorporated byreference.

Similarly as described before with respect to single-chain CARs, thepresent invention encompasses immune cells endowed with multi-chain CARswhich target specifically a cell surface marker such as CD38, CS1 orCD70. According to a preferred embodiment of the invention the CARsdescribed above are expressed in immune cells, whereas inactivation ofthe endogenous genes encoding said surface marker(s) is induced byexpression of a rare-cutting endonuclease.

Activation and Expansion of T Cells

The method according to the invention generally includes a further stepof activating and/or expanding the T-cells. This can be done prior to orafter genetic modification of the T cells, using the methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005. According to these methods, the T cells of the inventioncan be expanded by contact with a surface having attached thereto anagent that stimulates a CD3 TCR complex associated signal and a ligandthat stimulates a co-stimulatory molecule on the surface of the T cells.

In particular, T cell populations may be stimulated in vitro such as bycontact with an anti-CD3 antibody, or antigen-binding fragment thereof,or an anti-CD2 antibody immobilized on a surface, or by contact with aprotein kinase C activator (e.g., bryostatin) in conjunction with acalcium ionophore. For co-stimulation of an accessory molecule on thesurface of the T cells, a ligand that binds the accessory molecule isused. For example, a population of T cells can be contacted with ananti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T cells. To stimulateproliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3antibody and an anti-CD28 antibody. For example, the agents providingeach signal may be in solution or coupled to a surface. As those ofordinary skill in the art can readily appreciate, the ratio of particlesto cells may depend on particle size relative to the target cell. Infurther embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. Cell surface proteinsmay be ligated by allowing paramagnetic beads to which anti-CD3 andanti-CD28 are attached (3×28 beads) to contact the T cells. In oneembodiment the cells (for example, 4 to 10 T cells) and beads (forexample, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of1:1) are combined in a buffer, preferably PBS (without divalent cationssuch as, calcium and magnesium). Again, those of ordinary skill in theart can readily appreciate any cell concentration may be used. Themixture may be cultured for several hours (about 3 hours) to about 14days or any hourly integer value in between. In another embodiment, themixture may be cultured for 21 days. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4,1L-7, GM-CSF, -10, -2, 1L-15, TGFp, and TNF- or any other additives forthe growth of cells known to the skilled artisan. Other additives forthe growth of cells include, but are not limited to, surfactant,plasmanate, and reducing agents such as N-acetyl-cysteine and2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM,F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodiumpyruvate, and vitamins, either serum-free or supplemented with anappropriate amount of serum (or plasma) or a defined set of hormones,and/or an amount of cytokine(s) sufficient for the growth and expansionof T cells. Antibiotics, e.g., penicillin and streptomycin, are includedonly in experimental cultures, not in cultures of cells that are to beinfused into a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% C02). T-cells that havebeen exposed to varied stimulation times may exhibit differentcharacteristics.

In another particular embodiment, said cells can be expanded byco-culturing with tissue or cells. Said cells can also be expanded invivo, for example in the subject's blood after administrating said cellinto the subject.

Therapeutic Applications

The T-cells obtainable by the different methods described above areintended to be used as a medicament for treating, among others, cancer,infections or immune diseases in a patient in need thereof.

Said treatment can be ameliorating, curative or prophylactic. It may beeither part of an autologous immunotherapy or part of an allogenicimmunotherapy treatment. By autologous, it is meant that cells, cellline or population of cells used for treating patients are originatingfrom said patient or from a Human Leucocyte Antigen (HLA) compatibledonor. By allogeneic is meant that the cells or population of cells usedfor treating patients are not originating from said patient but from adonor.

The T-cells engineered according to one of the previous methods may bepooled, frozen, and administrated to one or several patients. When theyare made non-alloreactive, they are available as an “off the shelf”therapeutic product, which means that they can be universally infused topatients in need thereof.

Said treatments are primarily intended to patients diagnosed withcancer, viral infection, autoimmune disorders or Graft versus HostDisease (GvHD). Cancers are preferably leukemias and lymphomas, whichhave liquid tumors, but may also concern solid tumors. Types of cancersto be treated with the CARs of the invention include, but are notlimited to, carcinoma, blastoma, and sarcoma, and certain leukemia orlymphoid malignancies, benign and malignant tumors, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

The present invention provides in Tables 4 to 14 with examples ofantigen markers, which can be targeted with the engineered-cells of theinvention for treating different types of cancer.

Preferred antigen markers used for the immunotherapy of the presentinvention are more particularly CD38, CD319 (CS1) and CD70.

The present T-cells, when armed with specific CARs directed againstpatient's own immune cells, especially T-cells, allow the inhibition orregulation of said cells, which is a key step for treating auto-immunedisease, such as rheumatoid polyarthritis, systemic lupus erythematosus,Sjogren's syndrome, scleroderma, fibromyalgia, myositis, ankylosingspondylitis, insulin dependent diabetes of type I, Hashimoto'sthyroiditis, Addison's disease, Crohn's disease, Celiac's disease,amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS).Accordingly the present invention encompass a method for treating animmune disease by directing engineered T-cells as previously describedagainst patient's own T-cells.

The above treatments can take place in combination with one or moretherapies selected from the group of antibodies therapy, chemotherapy,cytokines therapy, dendritic cell therapy, gene therapy, hormonetherapy, laser light therapy and radiation therapy.

The engineered T-cells as previously described, when they are maderesistant to chemotherapy drugs and immunosuppressive drugs that areused as standards of care, especially methotrexate and the combinationof fludarabine and Cyclophosphamide, are particularly suited fortreating various forms of cancer. Indeed, the present inventionpreferably relies on cells or population of cells, In this aspect, it isexpected that the chemotherapy and/or immunosuppressive treatment shouldhelp the selection and expansion of the engineered T-cells in-vivo.

In certain embodiments of the present invention, cells are administeredto a patient in conjunction with (e.g., before, simultaneously orfollowing) any number of relevant treatment modalities, including butnot limited to treatment with agents such as antiviral therapy,cidofovir and interleukin-2, Cytarabine (also known as ARA-C) ornataliziimab treatment for MS patients or efaliztimab treatment forpsoriasis patients or other treatments for PML patients. In furtherembodiments, the T cells of the invention may be used in combinationwith chemotherapy, radiation, immunosuppressive agents, such ascyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAMPATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p7056 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, 1 1; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Citrr. Opin. mm n. 5:763-773, 93). In afurther embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH, In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery. Said modified cells obtained by any one of themethods described here can be used in a particular aspect of theinvention for treating patients in need thereof against Host versusGraft (HvG) rejection and Graft versus Host Disease (GvHD); therefore inthe scope of the present invention is a method of treating patients inneed thereof against Host versus Graft (HvG) rejection and Graft versusHost Disease (GvHD) comprising treating said patient by administering tosaid patient an effective amount of modified cells comprisinginactivated TCR alpha and/or TCR beta genes.

According to one embodiment, said T cells of the invention can undergorobust in vivo T cell expansion upon administration to a patient, andcan persist in the body fluids for an extended amount of time,preferably for a week, more preferably for 2 weeks, even more preferablyfor at least one month. Although the T-cells according to the inventionare expected to persist during these periods, their life span into thepatient's body are intended not to exceed a year, preferably 6 months,more preferably 2 months, and even more preferably one month.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The compositions described herein may beadministered to a patient subcutaneously, intradermaliy, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 10⁴-10⁹ cells per kg body weight, preferably 10⁵to 10⁶ cells/kg body weight including all integer values of cell numberswithin those ranges. The cells or population of cells can beadministrated in one or more doses. In another embodiment, saideffective amount of cells are administrated as a single dose. In anotherembodiment, said effective amount of cells are administrated as morethan one dose over a period time. Timing of administration is within thejudgment of managing physician and depends on the clinical condition ofthe patient. The cells or population of cells may be obtained from anysource, such as a blood bank or a donor. While individual needs vary,determination of optimal ranges of effective amounts of a given celltype for a particular disease or conditions within the skill of the art.An effective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administrated will be dependent uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment and the nature of the effectdesired.

In another embodiment, said effective amount of cells or compositioncomprising those cells are administrated parenterally. Saidadministration can be an intravenous administration. Said administrationcan be directly done by injection within a tumor.

Identification of Surface Antigen Marker Expressed on the Surface ofT-Cells, while being Overexpressed in Solid Tumors Involved intoDifferent Types of Cancer (Tables 5 to 13)

We used BioGPS microarray data from a panel of normal tissues (HumanU133A/GNF1H Gene Atlas) cancer microarray data that also can bedownloaded from BioGPS (Human Primary Tumors (U95)) uniprot data thatcontains the subcellular localization.

We drew the distribution of values coming from normal tissues anddetermined a threshold value of 5 for the relative expression.

We browsed all the genes assayed with microarrays (44.000 probesrepresenting about 13 000 genes) and checked their localization in themembrane (protein not referred to as being a membrane protein werediscarded). Expression in CD8+ T-cells was checked from the BioGPSdatabase. The genes were listed according to the type of cancer wherethe corresponding expression was the highest (Tables 5 to 13).

Identification of Surface Antigen Marker Expressed on the Surface ofT-Cells, while being Overexpressed in Different Liquid Blood Tumors(Table 14)

For that study, no RNA-seq data were available and thus we usedmicroarray data that were obtained from a large study from the MILEconsortium (Microarray Innovations in Leukemia), involving 11laboratories(http://www.ngrl.org.uk/wessex/downloads/tm08/TM08-54-1_KenMills.pdf—Haferlachet al. 2010, http://www.ncbi.nlm.nih.gov/pubmed/20406941). This raw datainclude results for ALL (acute lymphoblastic leukemia), AML (acutemyelogenous leukemia), CLL (chronic lymphoblastic leukemia) and CML(chronic myelogenous leukemia) and MDS (myelodysplastic syndrome). Wealso used uniprot data for subcellular localization as usual.

We first drew the overall distribution of values from all genes on allstudied tissues. Then, to have an idea of the level necessary forexpression, we took a list of genes which are expressed in some liquidtumors and for which therapeutic antibodies are available (CD52, CD 20,CD33, CD19, CD25, CD44, CD47, CD96, CD116, CD117, CD135, TIM-3). Foreach gene, we looked at the value obtained in the tumor in which it isexpressed. Then, we computed the average for each tumor and gene pairfor which the gene seems to give a cell membrane protein (cell membranelocalization+ description of at least one transmembrane domain in theprotein). We discarded genes for which the expression in all the tissueswas below this threshold of 0.15. We listed and ranked in Table 14,those genes which relative expression in T-cells was above 0.2. Thus,Table 4 provides putative antigen marker candidates for targeting liquidtumor cells as per the invention, in particular for treating ALL, AML,CLL, CML and MDS.

Example of Steps to Engineer T-Cells According to the Invention forImmunotherapy

For a better understanding of the invention, it is provided below anexample of the steps to follow to produce T-cells directed againstleukemia CD38 positive cells:

-   -   1. Providing T-cells from a cell culture or from a blood sample        from one individual patient or from blood bank and activating        said T cells using anti-CD3/C28 activator beads (Dynabeads®).        The beads provide both the primary and co-stimulatory signals        that are required for activation and expansion of T cells.    -   2. Transducing said cells with a retroviral vector comprising a        transgene encoding a Chimeric antigen receptor consisting of the        fusion of CD3zeta activation domain, 4-1BB co-stimulation        domain, a transmembrane domain and a hinge from CD28 fused to a        sequence encoding the variable chain of an anti-CD38 antibody.        For security improvement of the transformed T-cell, a suicide        gene sensitive to rituximab may further be introduced as        described in WO 2013/153391 into the lentiviral vector separated        by T2A splitting sequences.    -   3. (Optionally) Engineering non alloreactive and/or resistant T        cells:        -   a) It is possible to Inactivate TCR alpha in said cells to            eliminate the TCR from the surface of the cell and prevent            recognition of host tissue as foreign by TCR of allogenic            and thus to avoid GvHD by following the protocols set forth            in WO 2013/176915.        -   b) It is also possible to inactive one gene encoding target            for an immunosuppressive agent or a chemotherapy drug to            render said cells resistant to immunosuppressive or            chemotherapy treatment to prevent graft rejection without            affecting transplanted T cells. In this example, target of            immunosuppressive agents is CD52 and immunosuppressive agent            is a humanized monoclonal anti-CD52 antibody (ex:            Alemtuzumab) as described in WO 2013/176915.    -   4. Gene Inactivation is performed by electoporating T-cells with        mRNA encoding specific TAL-endonuclease (TALEN™—Cellectis, 8 rue        de la Croix Jarry, France). Inactivated T cells are sorted using        magnetic beads. For example, T cells still expressing the        targeted gene (e.g. CD38, CD70 and CD70) can be removed by        fixation on a solid surface, and inactivated cells are not        exposed of the stress of being passed through a column. This        gentle method increases the concentration of properly engineered        T-cells.    -   5. Expansion in vitro of engineered T-cells prior to        administration to a patient or in vivo following administration        to a patient through stimulation of CD3 complex. Before        administration step, patients can be subjected to an        immunosuppressive treatment such as CAMPATH1-H, a humanized        monoclonal anti-CD52 antibody.    -   6. Optionally exposed said cells with bispecific antibodies ex        vivo prior to administration to a patient or in vivo following        administration to a patient to bring the engineered cells into        proximity to a target antigen.

Functional Analysis of the Engineered T-Cells Electroporated with aMonocistronic mRNA Encoding for an Anti-CD38 Single Chain ChimericAntigen Receptor (CAR CD38):

To verify that genome engineering did not affect the ability of theengineered T-cells to present anti-tumor activity, especially whenprovided with a chimeric antigen receptor (CAR CD38), The engineeredT-cells were incubated for 4 hours with Daudi cells expressing CD38 ontheir surface. The cell surface upregulation of CD107a, a marker ofcytotoxic granule release by T lymphocytes (called degranulation) wasmeasured by flow cytometry analysis (Betts, Brenchley et al. 2003).

24 hours post electroporation, cells were stained with a fixableviability dye eFluor-780 and a PE-conjugated goat anti mouse IgG F(ab′)2fragment specific to assess the cell surface expression of the CAR onthe live cells. The vast majority of the live T-cells geneticallydisrupted for CD38, express the CAR on their surface. T cells wereco-cultured with Daudi (CD38⁺) cells for 6 hours and analyzed by flowcytometry to detect the expression of the degranulation marker CD107a attheir surface (Betts, Brenchley et al. 2003).

The results showed that CD38 disrupted T-cells kept the same ability todegranulate in response to PMA/ionomycin (positive control) or CD38+Daudi cells. CD107 upregulation is dependent on the presence of a CD38+.These data suggest that the genome engineering of the present T-cellshad no negative impact on the ability of T cells to mount a controlledanti-tumor response.

TABLE 4 Cluster of differentiation (CD) antigen markers of variouscancers found to be expressed on the surface of T-cells Antigen OtherNames Structure main Distribution Function CD1a T6 IgSF, MHC-likecortical thymocytes, Langerhans cells, DC antigen presentation, withbeta2m CD1b T6 IgSF, MHC-like cortical thymocytes, Langerhans cells, DCantigen presentation, with beta2m CD1c T6 IgSF, MHC-like corticalthymocytes, Langerhans cells, DC, B antigen presentation, with beta2msubset CD1d IgSF, MHC-like intestinal epith, B subset, monolow, DCantigen presentation, with beta2m CD3 gamma, T3 IgSF T, thymocyte subsetwith TCR, TCR surface expression/signal CD3 delta transduction CD3epsilon T3 IgSF T, thymocyte subset with TCR, TCR surfaceexpression/signal transduction CD4 T4 IgSF thymocyte subset, T subset,mono, mac MHC class II coreceptor, HIV receptor, T celldifferentiation/activation CD5 T1, Tp67 Scavenger R SF thymocytes, T, Bsubset, B-CLL CD72 receptor, TCR or BCR signaling, T-B interaction CD7IgSF hematopoietic progenitors, thymocytes, T, T costimulation NK CD8aT8, Leu-2 IgSF thymocyte subset, T subset, NK MHC class I coreceptor,receptor for some mutated HIV-1, T cell differentiation/activation CD8bIgSF thymocyte subset, T subset CD9 p24, MRP-1 TM4SF pre-B, eosinophils,basophils, platelets, Tact cellular adhesion and migration CD10 CALLA,NEP, type II TM B precursors, T precursors, neutrophils zinc-bindingmetalloproteinase, B cell gp100 development CD11a LFA-1, integrinIntegrin family lymph, gran, mono, mac CD11a/CD18 receptor for ICAM-1,-2, -3, alphaL intercellular adhesion, T costimulation CD11b Mac-1,integrin Integrin family myeloid cells, NK binds CD54, ECM, iC3b alphaMCD11c p150, 95, CR4, Integrin family DC, myeloid cells, NK, B, T subsetbinds CD54, fibrinogen and iC3b integrin alphaX CD13 Aminopeptidase typeII TM myeloid cells zinc-binding metalloproteinase, antigen N, APNprocessing, receptor for corona virus strains CD14 LPS-R GPI-linkedmono, mac, Langerhans cells, granlow receptor for LPS/LBP, LPSrecognition CD15 Lewis-x, Lex CHO neutrophils, eosinophils, monoadhesion CD16a FcgammaRIIIA IgSF neutrophils, mac, NK component of lowaffinity Fc receptor, phagocytosis and ADCC CD16b FcgammaRIIIB IgSFneutrophils component of low affinity Fc receptor, phagocytosis and ADCCCD20 B1, Bp35 TM4SF B, T subset B cell activation CD21 C3DR, CR2, CCRSFB, FDC, T subset complement C3d and EBV receptor, complex EBV-R withCD19 and CD81, BCR coreceptor CD22 BL-CAM, IgSF, B adhesion, B-mono, B-Tinteractions Siglec-2 sialoadhesins CD23 FcepsilonRII C-type lectin B,activated mac, eosinophils, FDC, platelets CD19-CD21-CD81 receptor, IgElow affinity receptor, signal transduction CD24 BA-1 GPI-linkedthymocytes, erythrocytes, peripheral lymph, binds P-selectin myeloidCD25 Tac, p55 type I TM Tact, Bact, lymph progenitors IL-2Ralpha, withIL-2Rbeta and gamma to form high affinity complex CD31 PECAM-1 IgSFmono, platelets, gran, endoth, lymph subset CD38 receptor, adhesion CD33p67, Siglec-3 IgSF, myeloid progenitors, mono, gran, DC, mast adhesionsialoadhesins cells, Tact CD37 TM4SF B, Tlow, granlow signaltransduction CD38 T10 variable levels on majority of hematopoieticecto-ADP-ribosyl cyclase, cell activation cells, high expression onplasma cells, B and Tact CD40 TNFRSF B, mono, mac, FDC, endoth, T subsetCD154 receptor, B differentiation/costimulation, isotype-switching,rescues B cells from apoptosis CD43 Leukosialin, Sialomucin, typeleukocytes, except resting B, plateletslow inhibition of T cellinteraction, CD54R, adhesion sialophorin I TM CD44 H-CAM, Pgp-1hyaladherin hematopoietic and non-hematopoietic cells, binds hyaluronicacid, adhesion family except platelets, hepatocytes, testis CD45 LCA,T200, hematopoietic cells, multiple isoforms from tyrosine phosphatase,enhanced TCR & BCR B220 alternative splicing signals CD45RA B, Tsubset(naive), mono exon A isoforms of CD45 CD45RB T subset, B, mono,mac, gran exon B isoforms of CD45 CD45RO Tact, memory T, B subset, mono,mac, gran isoform of CD45 lacking A, B, C exons CD46 MCP CCRSF nucleatedcells membrane cofactor protein, binds C3b & C4b allowing degradation byFactor I, measles virus receptor CD47 IAP IgSF hematopoietic cells,epith, endoth, fibroblasts, leukocyte adhesion, migration, activationother tissues CD48 Blast-1 IgSF broad, all leukocytes cell adhesion CD52CAMPATH-1 thymocytes, T, B (not plasma cells), mono, mac CD53 TM4SFleukocytes, DC, osteoblasts, osteoclasts signal transduction CD55 DAFGPI-linked hematopoietic, endoth binds C3b, complement regulation CD56NCAM IgSF NK, T subset, neurons, some large granular adhesion lymphocyteleukemias, myeloid leukemias CD57 HNK-1, Leu-7 NK subset, T subset CD58LFA-3 IgSF hematopoietic, non-hematopoietic cells CD2 receptor, adhesionCD59 Protectin, MAC- GPI-linked hematopoietic, non-hematopoietic cellsbinds complement C8 and C9, blocks assembly inhibitor of membrane attackcomplex CD60a GD3 CHO T subset, platelets, thymic epith, astrocytescostimulation CD63 LIMP, LAMP-3 TM4SF activated platelets, mono, maclysosomal membrane protein, moves to cell surface after activation CD68Macrosialin, Sialomucin intracellularly in mono, mac, neutrophils,basophils, large lymph, mast cells, gp110 DC, myeloid progenitors, liverCD69 AIM C-type lectin Tact, B, NK and gran, thymocytes, platelets,signal transduction Langerhans cells CD70 Ki-24 TNFSF Bact and Tact CD27ligand, T and B cell costimulation CD74 Ii, invariant B, mac, mono,Langerhans cells, DC, Tact MHC class II traffic and function chain CD79aIga IgSF B component of BCR, BCR surface expression and signaltransduction CD79b Igb IgSF B component of BCR, BCR surface expressionand signal transduction CD81 TAPA-1 TM4SF T, B, NK, thymocytes, DC,endoth, fibroblast, complex with CD19 & CD21, signaling, Tneuroblastomas, melanomas costimulation CD82 R2 TM4SF leukocytes signaltransduction CD83 HB15 IgSF Bact and Tact, DC, Langerhans cells CDw84mono, platelets, B, T subset, mac subset CD86 B70, B7-2 IgSF mono, DC,Bact and Tact binds to CD28, CD152, T costimulation CD87 UPA-RGPI-linked gran, mono, NK, Tact, endoth, fibroblasts urokinaseplasminogen activator receptor, inflammatory cell invasion, metastasisCD90 Thy-1 IgSF, GPI-linked CD34+ hematopoietic subset, neuronshematopoietic stem cell and neuron differentiation CD94 KP43 C-typelectin NK, T subset complex with NKG2, inhibits NK function CD95 Apo-1,Fas TNFRSF lymph (high upon activation), mono, FasL (CD178) receptor,apoptosis neutrophils CD96 TACTILE IgSF NK, Tact adhesion of activated Tand NK CD97 TM7SF Bact and Tact, mono, gran CD98 4F2 T, B, NK, gran, allhuman cell lines cellular activation CD99 MIC2, E2 leukocytes T cellactivation, adhesion CD100 hematopoietic cells except immature bone celladhesion, cellular activation marrow cells, RBC and platelets CD103HML-1, alpha6, Integrin family intraepithelial lymph, lymph subset,activated with integrin beta7, binds E-cadherin, lymph integrin alphaElymph homing/retention CD107a LAMP-1 activated platelets, T, endoth,metastatic tumors a lysosomal membrane protein CD107b LAMP-2 activatedplatelets, T, endoth, metastatic tumors a lysosomal membrane proteinCD109 Tact and platelets, CD34+ subset, endoth CD123 IL-3R CRSF lymphsubset, basophils, hematopoietic IL-3Ralpha, with CDw131 progenitors,mac, DC, megakaryocytes CD146 MUC18, S-endo IgSF endoth, melanomas, FDC,Tact adhesion CD154 CD40L, gp39, TNFSF Tact CD40 ligand, B and DCcostimulation TRAP CD158a p58.1 IgSF, KIR family NK subset, T subsetinhibition of NK cell cytolytic activity, MHC class-I specific NKreceptor CD158b p58.2 IgSF, KIR family NK subset, T subset inhibition ofNK cell cytolytic activity, MHC class-I specific NK receptor CD163 130kD Scavenger mono, mac receptor SF CD164 MGC-24 epith, mono,hematopoietic progenitor cell-stromal cell interaction lymphlow, bonemarrow stromal cells, CD34+ erythroid progenitors CD168 RHAMM mono, Tsubset, thymocyte subset, adhesion, tumor migration, metastasisintracellularly in breast cancer cells CD171 L1 IgSF CNS, PNS, glialcells, mono, T subset, B, DC, kidney morphogenesis, lymph nodearchitecture, several human tumor cells T costimulation,neurohistogenesis, homotypic interaction, binds CD9, CD24, CD56, CD142,CD166, integrins CD177 NB1 neutrophil subset CD178 FasL, CD95L TNFSFTact, testis CD95 ligand, apoptosis, immune privilege, soluble form inserum CD180 RP-105 LRRF, TLR B subset, mono, DC B cell activation, LPSsignaling, with MD-1 family CD182 CXCR2, IL-8RB GPCR1 familyneutrophils, basophils, NK, T subset, mono binding of IL-8 induceschemotaxis of neutrophils CD185 CXCR5, BLR1 GPCR1 family mature B andBurkitt Lymphoma cells with chemokine BLC, possible regulatory functionin Burkitt Lymphomagenesis and/or B differentiation, activation ofmature B CD191 CCR1, MIP- GPCR1 family T, mono, stem cell subset bindsC-C type chemokines and transduces 1alphaR, signal by increasingintracellular calcium ion RANTES-R levels CD193 CCR3, CKR3 GPCR1 familyeosinophils, lower expression in neutrophils binds eotaxin, eotaxin-3,MCP-3, MCP-4, and mono, T subset RANTES & MIP-1 delta, alternativecoreceptor with CD4 for HIV-1 infectiongg CD196 CCR6, LARC GPCR1 familyT subset, B, DC subset binds MIP-3alpha/LARC receptor, DRY6 CD197 CCR7 Tsubset, DC Subset 6Ckine and MIP-2beta receptor CD200 OX-2 thymocytes,endoth, B, Tact inhibition of immune response CD209 DC-SIGN DC subsetICAM-3 receptor, HIV-1 binding protein CD227 MUC1, EMA Mucin family,epith, stem cell subset, FDC, mono, B subset, adhesion, signaling, bindsCD169, CD54, & type I TM some myelomas selectins CD231 TALLA-1, A15TM4SF T leukemias, neuroblastomas, brain neurons marker for T cell acutelymphoblastic leukemia CD246 ALK, Ki-1 anaplastic T cell leukemias,small intestine, brain development, implicated in ALK testis, brain, noton normal lymph lymphomas CD254 TRANCE, TNFSF lymph node & BM stromaTact binds OPG and RANK, osteoclast differentiation, RANKL, OPGLenhances DC to stimulate naïve-T proliferation CD263 TRAIL-R3,peripheral blood lymphocytes receptor for TRAIL but lacks death domainDcR1, LIT CD272 BTLA IgSF Tact, B, remains on Th1 HVEM receptor,inhibitory response CD273 B7DC, PD-L2, IgSF DC subset, mono, mac PD-1receptor, costimulation or suppression of T PDCD1L2 proliferation CD276B7-H3 B7 Family, ASV in vitro cultured DC and mono, Tact, mammarycostimulation, T activation tissue CD277 BT3.1, B7/BT family, T, B, NK,mono, DC, endoth, CD34+ cells, T activation butyrophilin ASV tumor celllines SF3 A1, BTF5 CD279 PD1, SLEB2 Tact and Bact B7-H1 & B7-DCreceptor, autoimmune disease and peripheral tolerance CD298 Na+/K+−broad transport sodium & potassium ions across ATPase beta3 membranesubunit CD300a CMRF35H, IgSF, ASV NK, mono, neutrophils, T and B subsetand unknown IRC1, IRp60 lymphocytic cell lines, AML CD300c CMRF35A, LIRIgSF mono, neutrophils, monocytic cell lines, B & T unknown subsetsCD304 BDCA4, semaphorin neurons, CD4+/CD25+ Treg, DC, endothelialinteracts with VEGF165 & semaphorins, co- neuropilin 1 family and tumorcells receptor with plexin, axonal guidance, angiogenesis, cellsurvival, migration CD305 LAIR1 IgSF, ASV NK, B, T, mono inhibitoryreceptor on NK and T cells CD314 NKG2D, KLR Type II lectin-like NK, CD8+activated, NK1.1+ T, some myeloid binds MHC class I, MICA, MICB, Rae1 &receptor cells ULBP4, activates cytolysis and cytokine production,costimulation CD317 BST2, HM1.24 Type II B, T, NK, mono, DC, fibroblastcell line, pre-B cell growth, overexpressed in multiple myeloma myelomaCD319 CS1, CRACC, SLAM receptor B Cells, Dendritic Cells, NK, NKTmultiple myeloma SLAMF7 family

TABLE 5 antigen markers expressed on the surface of both colon tumorcells and T-cells Relative Relative Expression expression in colonAntigen Protein Name in T-Cell cancer cells EPCAM Epithelial celladhesion molecule 2.97 13.99 IFITM1 Interferon-induced transmembraneprotein 1 10.55 13.06 CLDN4 Claudin-4 2.87 11.62 CDH17 Cadherin-17 1.8511.52 CEACAM1 Carcinoembryonic antigen-related cell adhesion molecule 13.33 10.84 SLC26A3 Chloride anion exchanger 2.57 10.59 ATP1A1Sodium/potassium-transporting ATPase subunit alpha-1 9.28 10.51 SIIsomaltase 2.86 10.46 ABCB1 Multidrug resistance protein 1 6.09 10.24KCNQ1 Potassium voltage-gated channel subfamily KQT member 1 3.36 9.99FCGRT IgG receptor FcRn large subunit p51 4.8 9.98 EPHB3 Ephrin type-Breceptor 3 5.23 9.74 DSG2 Desmoglein-2 3.04 8.5 EPHB4 Ephrin type-Breceptor 4 6.5 8.44 GUCY2C Heat-stable enterotoxin receptor 2.23 8.05EPHA2 Ephrin type-A receptor 2 2.8 7.95 LY6G6D Lymphocyte antigen 6complex locus protein G6f 2.02 7.91 CD97 CD97 antigen subunit beta 7.77.87 SIGMAR1 Sigma non-opioid intracellular receptor 1 4.58 7.85 EREGEpiregulin 2.93 6.9 FAIM2 Protein lifeguard 2 2.94 6.82 PIGR Secretorycomponent 4.2 6.8 SLC7A6 Y + L amino acid transporter 2 8.06 6.55 SCNN1DAmiloride-sensitive sodium channel subunit delta 1.77 5.74 GPR35G-protein coupled receptor 35 1.98 5.5 ABCG2 ATP-binding cassettesub-family G member 2 1.79 5.35 LPAR4 Lysophosphatidic acid receptor 42.93 5.05 GPR161 G-protein coupled receptor 161 2.71 4.96 CD1C T-cellsurface glycoprotein CD1c 2.73 4.89 SGCA Alpha-sarcoglycan 2.32 4.84CD22 B-cell receptor CD22 4.12 4.75 CD22 B-cell receptor CD22 3.58 4.75CD22 B-cell receptor CD22 2.73 4.75 CD22 B-cell receptor CD22 2.14 4.75SLC22A18 Solute carrier family 22 member 18 2.32 4.62 HTR75-hydroxytryptamine receptor 7 3.02 4.46 LCT Phlorizin hydrolase 2.324.24 CD33 Myeloid cell surface antigen CD33 3.42 4.14 PVR Poliovirusreceptor 5.07 4.07 PLXDC1 Plexin domain-containing protein 1 5.85 3.99P2RY2 P2Y purinoceptor 2 2.15 3.97 CHRNB2 Neuronal acetylcholinereceptor subunit beta-2 6.31 3.88 PTGDR Prostaglandin D2 receptor 4.083.65 NCR1 Natural cytotoxicity triggering receptor 1 2.63 3.33 GYPAGlycophorin-A 3.18 3.31 TNFRSF8 Tumor necrosis factor receptorsuperfamily member 8 2 2.75 KEL Kell blood group glycoprotein 1.93 2.48EDA Ectodysplasin-A, secreted form 2.7 2.42 ACE Angiotensin-convertingenzyme, soluble form 2.39 2.19 DRD2 D(2) dopamine receptor 2.49 1.97CXCR3 C-X-C chemokine receptor type 3 4.19 1.66 MC2R Adrenocorticotropichormone receptor 1.94 1.43

TABLE 6 antigen markers expressed on the surface of both breast tumorcells and T-cells Relative Relative Expression expression in colonAntigen Protein Name in T-Cell cancer cells ABCA8 ATP-binding cassettesub-family A member 8 3.15 7.73 ABCC10 Multidrug resistance-associatedprotein 7 6.48 5.29 ABCC6 Multidrug resistance-associated protein 6 2.672.17 ACCN2 Acid-sensing ion channel 1 3.62 2.49 ADAM12 Disintegrin andmetalloproteinase domain-containing 4.96 7.72 protein 12 ADCYAP1R1Pituitary adenylate cyclase-activating polypeptide type I 2.17 2.88receptor ADRA1A Alpha-1A adrenergic receptor 3.31 4.85 ADRA1B Alpha-1Badrenergic receptor 1.49 1.6 ADRA1D Alpha-1D adrenergic receptor 2.393.38 ADRA2A Alpha-2A adrenergic receptor 2.64 1.79 ADRB3 Beta-3adrenergic receptor 2.36 2.16 AGER Advanced glycosylation endproduct-specific receptor 2.85 2.38 AGTR2 Type-2 angiotensin II receptor3.08 3.7 ALK ALK tyrosine kinase receptor 4.97 4.27 ANO3 Anoctamin-32.39 3.69 ANPEP Aminopeptidase N 3.26 10.78 APLNR Apelin receptor 2.472.06 AQP2 Aquaporin-2 2.12 1.43 ATP10A Probablephospholipid-transporting ATPase VA 3.96 6.02 ATP2B2 Plasma membranecalcium-transporting ATPase 4 2.75 4.81 ATP2B3 Plasma membranecalcium-transporting ATPase 3 3.7 4.14 ATP4A Potassium-transportingATPase alpha chain 1 1.56 11.49 ATP4B Potassium-transporting ATPasesubunit beta 2.49 13.56 ATP6V0A2 V-type proton ATPase 116 kDa subunit aisoform 2 2.51 2.57 ATRN Attractin 4.09 9.44 AVPR1A Vasopressin V1areceptor 2.52 4.03 AVPR1B Vasopressin V1b receptor 2.97 3.32 AVPR2Vasopressin V2 receptor 2.68 2.93 BAI1 Brain-specific angiogenesisinhibitor 1 2.73 0.33 BAI2 Brain-specific angiogenesis inhibitor 2 2.344.14 BAI3 Brain-specific angiogenesis inhibitor 3 2.73 4.76 BDKRB1 B1bradykinin receptor 2.07 3.28 BRS3 Bombesin receptor subtype-3 2.74 4.12BTF3 Butyrophilin subfamily 3 member A2 11.29 13.02 C18orf1 Low-densitylipoprotein receptor class A domain-containing 3.18 8.45 protein 4 C3AR1C3a anaphylatoxin chemotactic receptor 3.04 5.15 C6orf105Androgen-dependent TFPI-regulating protein 2.34 3.84 CASR Extracellularcalcium-sensing receptor 2.52 5 CCBP2 Atypical chemokine receptor 2 1.723.29 CCKAR Cholecystokinin receptor type A 2.46 3 CCKBRGastrin/cholecystokinin type B receptor 2.25 5.66 CCR2 C-C chemokinereceptor type 2 5.94 3.56 CCR3 C-C chemokine receptor type 3 1.89 4.17CCR6 C-C chemokine receptor-like 2 3.33 5.23 CCR8 C-C chemokine receptortype 8 2.28 3.93 CCR9 C-C chemokine receptor type 9 1.68 1.98 CD1AT-cell surface glycoprotein CD1a 1.98 4.88 CD1B T-cell surfaceglycoprotein CD1b 2.35 4.94 CD1D Antigen-presenting glycoprotein CD1d2.82 4.96 CD300C CMRF35-like molecule 6 2.04 5.04 CD4 T-cell surfaceglycoprotein CD4 2.84 6.17 CD40LG CD40 ligand, soluble form 2.1 3.49 CD5T-cell surface glycoprotein CD5 3.14 1.01 CD63 CD63 antigen 8.6 13.18CD84 SLAM family member 5 4.7 3.17 CDH15 Cadherin-15 2.07 3.55 CDH19Protocadherin-16 2.82 8.4 CDH22 Cadherin-22 3 4.9 CDH8 Cadherin-8 3.635.87 CDON Cell adhesion molecule-related/down-regulated by 2.35 3.61oncogenes CHRNA4 Neuronal acetylcholine receptor subunit alpha-4 2.143.33 CHRNA5 Neuronal acetylcholine receptor subunit alpha-5 2.2 4.88CHRNA6 Neuronal acetylcholine receptor subunit alpha-6 2.26 4.93 CHRNB3Neuronal acetylcholine receptor subunit beta-3 1.85 3.91 CHRNEAcetylcholine receptor subunit epsilon 2.56 2.83 CLDN3 Claudin-3 2.9113.56 CLDN7 Claudin-7 1.89 12.87 CLDN8 Claudin-8 2.46 10.67 CLDN9Claudin-9 1.74 1.69 CLEC4M C-type lectin domain family 4 member M 2.73.32 CMKLR1 Chemokine-like receptor 1 2.62 5 CNNM2 Metal transporterCNNM2 2.47 5.32 CNR2 Cannabinoid receptor 2 2.38 3.66 CRHR1Corticotropin-releasing factor receptor 1 2.15 10.71 CRHR2Corticotropin-releasing factor receptor 2 2.32 6.44 CSF1 Processedmacrophage colony-stimulating factor 1 5.63 7.61 CSF1R Macrophagecolony-stimulating factor 1 receptor 2.2 4.02 CSF3R Granulocytecolony-stimulating factor receptor 1.85 2.8 CX3CL1 Processed fractalkine2.35 9.31 CXCR5 C-X-C chemokine receptor type 5 2.07 6.06 DAGLASn1-specific diacylglycerol lipase alpha 2.6 2.11 DRD1 D(1A) dopaminereceptor 2.67 5.71 DRD3 D(3) dopamine receptor 2.72 4.99 DRD4 D(4)dopamine receptor 1.49 0.89 DRD5 D(1B) dopamine receptor 2.26 4.91 DSC2Desmocollin-2 2.26 11.12 DSCAM Down syndrome cell adhesion molecule 2.543.76 DSG1 Desmoglein-1 2.62 7.71 EMR2 EGF-like module-containingmucin-like hormone receptor- 2.25 3.38 like 2 EPHA5 Ephrin type-Areceptor 5 2.42 7.48 EPHA7 Ephrin type-A receptor 7 2.61 4.87 ERBB3Receptor tyrosine-protein kinase erbB-3 2.39 12.76 F2RL2Proteinase-activated receptor 3 3.2 5.16 FAM168B Myelin-associatedneurite-outgrowth inhibitor 8.34 11.16 FAP Seprase 1.87 10.15 FAS Tumornecrosis factor receptor superfamily member 6 5.68 7.24 FASLG FasLintracellular domain 2.23 2.66 FCAR Immunoglobulin alpha Fc receptor 2.83.85 FCER1A High affinity immunoglobulin epsilon receptor subunit alpha2.54 4.59 FCGR2A Low affinity immunoglobulin gamma Fc region receptorII-a 2.77 8.81 FCGR2B Low affinity immunoglobulin gamma Fc regionreceptor II-b 2.46 5.35 FGFR2 Fibroblast growth factor receptor 2 4.019.83 FGFR4 Fibroblast growth factor receptor 4 2.56 7.42 FLT3LGFms-related tyrosine kinase 3 ligand 7.86 4.37 FPR1 fMet-Leu-Phereceptor 3.38 5.92 FPR3 N-formyl peptide receptor 3 1.91 2.61 FSHRFollicle-stimulating hormone receptor 1.89 3.78 FZD5 Frizzled-5 2.82 5.2FZD5 Frizzled-5 1.81 5.2 FZD9 Frizzled-9 2.66 3.16 GABRA1Gamma-aminobutyric acid receptor subunit alpha-1 2.2 6.26 GABRA5Gamma-aminobutyric acid receptor subunit alpha-5 2.49 3.24 GABRA6Gamma-aminobutyric acid receptor subunit alpha-6 2.54 2.98 GABRB1Gamma-aminobutyric acid receptor subunit beta-1 1.89 2.37 GABRB2Gamma-aminobutyric acid receptor subunit beta-2 2.26 3.89 GABRG3Gamma-aminobutyric acid receptor subunit gamma-3 2.23 2.85 GABRPGamma-aminobutyric acid receptor subunit pi 2.93 12.34 GABRR1Gamma-aminobutyric acid receptor subunit rho-1 2.35 3.47 GABRR2Gamma-aminobutyric acid receptor subunit rho-2 4.16 5.43 GALR2 Galaninreceptor type 2 1.85 0.46 GALR3 Galanin receptor type 3 0.68 0.48 GCGRGlucagon receptor 1.38 3.4 GHRHR Growth hormone-releasing hormonereceptor 1.61 3.49 GJA5 Gap junction alpha-5 protein 1.72 2.05 GJA8 Gapjunction alpha-8 protein 2.39 6.51 GJC1 Gap junction delta-3 protein1.94 3.89 GLP1R Glucagon-like peptide 1 receptor 5.72 3.41 GLRA1 Glycinereceptor subunit alpha-1 2.15 3.87 GLRA3 Glycine receptor subunitalpha-3 3.19 3.1 GNRHR Gonadotropin-releasing hormone receptor 2.72 4.1GPNMB Transmembrane glycoprotein NMB 2.14 13.94 GPR1 G-protein coupledreceptor 1 3.83 4.1 GPR135 Probable G-protein coupled receptor 135 4.151.91 GPR143 G-protein coupled receptor 143 1.93 3.65 GPR15 G-proteincoupled receptor 15 1.81 4.41 GPR17 Uracil nucleotide/cysteinylleukotriene receptor 1.93 1.74 GPR171 Probable G-protein coupledreceptor 171 7.73 6.32 GPR18 N-arachidonyl glycine receptor 7.05 3.52GPR182 G-protein coupled receptor 182 1.66 1.29 GPR19 Probable G-proteincoupled receptor 19 1.89 5.26 GPR20 G-protein coupled receptor 20 2.022.53 GPR3 G-protein coupled receptor 3 3.01 5.36 GPR3112-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid receptor 1.63 1.64GPR37L1 Prosaposin receptor GPR37L1 2.23 4 GPR39 G-protein coupledreceptor 39 1.81 1.36 GPR44 Prostaglandin D2 receptor 2 2 2.32 GPR45Probable G-protein coupled receptor 45 2.78 5.31 GPR6 G-protein coupledreceptor 6 2.56 3.38 GPR65 Psychosine receptor 6.59 4.5 GPR68 Ovariancancer G-protein coupled receptor 1 2.12 1.09 GPR98 G-protein coupledreceptor 98 1.89 4.7 GRIA1 Glutamate receptor 1 4.17 4.77 GRIA3Glutamate receptor 3 2.51 6.83 GRIK2 Glutamate receptor ionotropic,kainate 5 2.56 4.94 GRIK3 Glutamate receptor ionotropic, kainate 3 2.053.58 GRIN1 Glutamate receptor ionotropic, NMDA 1 4.52 1.49 GRIN2BGlutamate receptor ionotropic, NMDA 2B 2.22 3.56 GRIN2C Glutamatereceptor ionotropic, NMDA 2C 2.56 3.37 GRM1 Metabotropic glutamatereceptor 1 3.21 3.69 GRM2 Metabotropic glutamate receptor 2 2.04 0.44GRM3 Metabotropic glutamate receptor 3 2.39 3.41 GRM4 Metabotropicglutamate receptor 4 5.2 3.78 GRM5 Metabotropic glutamate receptor 52.26 5.28 GRM7 Metabotropic glutamate receptor 7 2.86 3.07 GYPBGlycophorin-B 2.43 4.02 HBP1 Glycosylphosphatidylinositol-anchored highdensity 7.32 9.27 lipoprotein-binding protein 1 HCRTR2 Orexin receptortype 2 2.32 2.42 HTR1B 5-hydroxytryptamine receptor 1B 2.82 3.51 HTR1D5-hydroxytryptamine receptor 1D 2.29 2.33 HTR1E 5-hydroxytryptaminereceptor 1E 1.72 2.4 HTR2A 5-hydroxytryptamine receptor 2A 2.1 3.67HTR2C 5-hydroxytryptamine receptor 2C 2.49 5.18 HTR4 5-hydroxytryptaminereceptor 4 3.86 4.25 ICAM4 Intercellular adhesion molecule 4 2.51 2.16ICOS Inducible T-cell costimulator 3.91 3.86 IL6R Interleukin-6 receptorsubunit alpha 4.24 3.08 IL6R Interleukin-6 receptor subunit alpha 2.643.08 IL6ST Interleukin-6 receptor subunit beta 9.43 12.67 IL9RInterleukin-9 receptor 2.71 2.86 ITGB3 Integrin beta-3 4.16 3.69 KCNA3Potassium voltage-gated channel subfamily A member 3 2.09 4.9 KCND2Potassium voltage-gated channel subfamily D member 2 2.67 4.25 KCNH1Potassium voltage-gated channel subfamily H member 1 2.31 4.48 KCNJ4Inward rectifier potassium channel 4 2.43 3.49 KCNMA1 Calcium-activatedpotassium channel subunit alpha-1 2.35 7.17 KCNS1 Potassiumvoltage-gated channel subfamily S member 1 5.66 6.49 KCNV2 Potassiumvoltage-gated channel subfamily V member 2 2.38 4.06 KIR2DL4 Killer cellimmunoglobulin-like receptor 2DL4 1.68 3.31 KIR3DL1 Killer cellimmunoglobulin-like receptor 3DL1 2.56 2.73 KIR3DL3 Killer cellimmunoglobulin-like receptor 3DL3 1.7 3.06 KLRG1 Killer cell lectin-likereceptor subfamily G member 1 8.3 5.76 LAMP1 Lysosome-associatedmembrane glycoprotein 1 10.9 13.6 LHCGR Lutropin-choriogonadotropichormone receptor 2.23 4.92 LNPEP Leucyl-cystinyl aminopeptidase,pregnancy serum form 2.68 5.05 LPAR2 Lysophosphatidic acid receptor 25.5 4.23 LRIG2 Leucine-rich repeats and immunoglobulin-like domains 3.355.48 protein 2 LRRTM2 Leucine-rich repeat transmembrane neuronal protein2 2.42 4.24 LTB4R Leukotriene B4 receptor 1 4.96 2.26 MAS1Proto-oncogene Mas 1.91 3.11 MC1R Melanocyte-stimulating hormonereceptor 2.94 0.96 MC5R Melanocortin receptor 5 2.28 1.63 MEP1B Meprin Asubunit beta 2.61 3.87 MFSD5 Molybdate-anion transporter 1.98 4.72 MOGMyelin-oligodendrocyte glycoprotein 3.08 4.74 MTNR1B Melatonin receptortype 1B 1.61 1.67 MUC1 Mucin-1 subunit beta 2.73 13.68 MUSK Muscle,skeletal receptor tyrosine-protein kinase 2.39 4.75 NCAM2 Neural celladhesion molecule 2 2.12 4.49 NCR2 Natural cytotoxicity triggeringreceptor 2 4.79 7.09 NCR3 Natural cytotoxicity triggering receptor 34.55 2.74 NIPA2 Magnesium transporter NIPA2 6.77 3.9 NLGN1 Neuroligin-12.62 7.71 NLGN4Y Neuroligin-4, Y-linked 2.52 5.26 NMBR Neuromedin-Breceptor 1.68 2.47 NPHS1 Nephrin 2.74 4.33 NPY2R Neuropeptide Y receptortype 2 2.68 4.43 NPY5R Neuropeptide Y receptor type 5 2.38 5.05 NTSR2Neurotensin receptor type 2 1.72 3 OPRD1 Delta-type opioid receptor 2.262.14 OPRL1 Nociceptin receptor 2.31 1.51 OPRM1 Mu-type opioid receptor3.18 4.01 OR10H3 Olfactory receptor 10H3 1.63 4.02 OR1E1 Olfactoryreceptor 1E1 3.04 4.77 OR2F1 Olfactory receptor 2F1 2.64 5.73 OR2F2Olfactory receptor 2F2 2.19 2.3 OR2H1 Olfactory receptor 2H1 3.39 3.82OR2H2 Olfactory receptor 2H2 3.79 6.37 OR2J2 Olfactory receptor 2J2 2.412.16 OR2J2 Olfactory receptor 2J2 1.93 2.16 OR5I1 Olfactory receptor 5I11.85 2.8 OR7E24 Olfactory receptor 7E24 2.5 3.47 P2RX7 P2X purinoceptor7 2.36 2.15 PANX1 Pannexin-1 2.14 4.38 PCDHA9 Protocadherin alpha-9 2.823.56 PCDHB11 Protocadherin beta-11 1.91 5.23 PCDHGA8 Protocadheringamma-A8 3.13 4.48 PLA2R1 Soluble secretory phospholipase A2 receptor2.91 5.16 PLXNA3 Plexin-A3 2.42 3.25 POP1 Blood vessel epicardialsubstance 1.74 2.59 PPYR1 Neuropeptide Y receptor type 4 2.2 2.75 PTGER1Prostaglandin E2 receptor EP1 subtype 1.96 0.94 PTGFR ProstaglandinF2-alpha receptor 2.75 4.89 PTGIR Prostacyclin receptor 2.78 2.12 PTPRJReceptor-type tyrosine-protein phosphatase eta 2.63 4.6 PTPRRReceptor-type tyrosine-protein phosphatase R 2.47 9.99 PVRL1 Poliovirusreceptor-related protein 1 2.52 4.51 PVRL2 Poliovirus receptor-relatedprotein 2 3.84 10.05 ROS1 Proto-oncogene tyrosine-protein kinase ROS2.93 3.38 S1PR2 Sphingosine 1-phosphate receptor 2 1.74 1.17 S1PR4Sphingosine 1-phosphate receptor 4 4 0.21 SCNN1B Amiloride-sensitivesodium channel subunit beta 1.89 3.16 SCNN1G Amiloride-sensitive sodiumchannel subunit gamma 2.23 2.61 SEMA4D Semaphorin-4D 10.66 1.56 SEMA6ASemaphorin-6A 4.55 7.81 SEMA6C Semaphorin-6C 5.02 3.73 SGCBBeta-sarcoglycan 2.69 3.45 SGCB Beta-sarcoglycan 2.04 3.45 SLC12A3Solute carrier family 12 member 3 2.26 3.36 SLC14A1 Urea transporter 12.97 6.21 SLC14A2 Urea transporter 2 2.85 4.4 SLC16A1 Monocarboxylatetransporter 1 3.46 8.84 SLC16A2 Monocarboxylate transporter 8 1.77 5.17SLC16A6 Monocarboxylate transporter 7 2.41 11.66 SLC22A1 Solute carrierfamily 22 member 1 2.95 11.61 SLC22A6 Solute carrier family 22 member 62.26 2.53 SLC5A12 Sodium-coupled monocarboxylate transporter 2 2.98 4.45SLC6A1 Sodium- and chloride-dependent GABA transporter 1 2.45 4.3 SLC6A4Sodium-dependent serotonin transporter 2.17 2.66 SLC6A6 Sodium- andchloride-dependent taurine transporter 2.54 4.13 SLC7A7 Y + L amino acidtransporter 1 2.22 9.78 SLC8A1 Sodium/calcium exchanger 1 2.07 2.36SLC9A1 Sodium/hydrogen exchanger 1 3.15 5.54 SLC9A3 Sodium/hydrogenexchanger 3 2.12 3.15 SLCO1A2 Solute carrier organic anion transporterfamily member 1A2 3.87 4.98 SLCO2B1 Solute carrier organic aniontransporter family member 2B1 4.43 8.92 SORT1 Sortilin 2.93 4.6 SSTR2Somatostatin receptor type 2 3.08 4.47 SSTR3 Somatostatin receptor type3 2.23 1.5 SSTR4 Somatostatin receptor type 4 1.83 1.53 SSTR5Somatostatin receptor type 5 2.57 1.47 TACR1 Substance-P receptor 2.663.2 TACR3 Neuromedin-K receptor 2.32 5.7 TLR6 Toll-like receptor 6 2.24.58 TMPRSS6 Transmembrane protease serine 6 4.02 3.69 TNFSF11 Tumornecrosis factor ligand superfamily member 11, 2.57 5.18 TNFSF14 Tumornecrosis factor ligand superfamily member 14, 3.34 2.83 soluble form TPOThyroid peroxidase 1.96 1.89 TRAT1 T-cell receptor-associatedtransmembrane adapter 1 7.51 5.29 TRHR Thyrotropin-releasing hormonereceptor 2 4.18 TRPM1 Transient receptor potential cation channelsubfamily M 2.43 5.22 member 1 TSHR Thyrotropin receptor 2.9 4.87 TSHRThyrotropin receptor 2.12 4.87 UNC93A Protein unc-93 homolog A 2.64 4.94VIPR2 Vasoactive intestinal polypeptide receptor 2 2.58 3.37 ZP2Processed zona pellucida sperm-binding protein 2 1.94 3.55

TABLE 7 antigen markers expressed on the surface of both digestive tumorcells and T-cells Relative Relative Expression expression in colonAntigen Protein Name in T-Cell cancer cells ACVR1B Activin receptortype-1B 5.16 10.48 AMIGO2 Amphoterin-induced protein 2 6.73 8.2 ATP1B1Sodium/potassium-transporting ATPase subunit beta-1 2.64 12.31 ATP8B1Probable phospholipid-transporting ATPase IC 8.22 2.17 CCR7 C-Cchemokine receptor type 7 10.25 11.52 CD164 Sialomucin core protein 2410.27 12.12 CD180 CD180 antigen 2.5 6.47 CD40 Tumor necrosis factorreceptor superfamily member 5 5.02 6 CD53 Leukocyte surface antigen CD5310.79 11.3 CD79A B-cell antigen receptor complex-associated proteinalpha 3.74 9.17 chain CD79B B-cell antigen receptor complex-associatedprotein beta 3.6 6.66 chain CD8B T-cell surface glycoprotein CD8 betachain 8.43 2.62 CELSR1 Cadherin EGF LAG seven-pass G-type receptor 12.72 8.68 CLCN5 H(+)/Cl(−) exchange transporter 5 2.71 4.97 CLDN18Claudin-18 3.05 14.51 CLIC1 Chloride intracellular channel protein 19.94 13.83 COL13A1 Collagen alpha-1(XIII) chain 2.96 6.24 DIO3 Type IIIiodothyronine deiodinase 2.04 2.9 EDNRA Endothelin-1 receptor 2.9 8.96EMR1 EGF-like module-containing mucin-like hormone receptor- 1.83 7.29like 1 ENPP1 Nucleotide pyrophosphatase 2.57 9.66 EPHB1 Ephrin type-Breceptor 1 2.02 6.33 EPHB1 Ephrin type-B receptor 1 1.81 6.33 F2RProteinase-activated receptor 1 3.04 9.78 F2RL1 Proteinase-activatedreceptor 2, alternate cleaved 2 3.31 9.47 FCER2 Low affinityimmunoglobulin epsilon Fc receptor soluble 2.49 8.77 form GABBR1Gamma-aminobutyric acid type B receptor subunit 1 5.1 8.52 GABRA3Gamma-aminobutyric acid receptor subunit alpha-3 2.12 3.84 GPR183G-protein coupled receptor 183 4.79 10.22 GPR37 Prosaposin receptorGPR37 3.1 8.23 GPRC5A Retinoic acid-induced protein 3 1.87 13.69 GRPRGastrin-releasing peptide receptor 2.04 3.35 GYPC Glycophorin-C 9.227.58 IL1R2 Interleukin-1 receptor type 2, soluble form 2.82 12.83KIAA0319 Dyslexia-associated protein KIAA0319 2.43 5.61 LAMP2Lysosome-associated membrane glycoprotein 2 4.05 11.29 LRP8 Low-densitylipoprotein receptor-related protein 8 4.24 8.84 LSRLipolysis-stimulated lipoprotein receptor 4.99 11.48 MICB MHC class Ipolypeptide-related sequence B 5.27 9.89 MMP16 Matrixmetalloproteinase-16 3.19 6.18 MS4A1 B-lymphocyte antigen CD20 2.15 8.02MYOF Myoferlin 2.41 11.56 NAT1 Sodium-coupled neutral amino acidtransporter 3 3.49 12.09 NFASC Neurofascin 3.78 8.28 NPY1R NeuropeptideY receptor type 1 2.32 6.93 OR2B6 Olfactory receptor 2B6 2.78 4.24P2RY10 Putative P2Y purinoceptor 10 3.39 6.62 PCDH1 Protocadherin-1 4.4510.07 PROM1 Prominin-1 2.52 11.77 PSEN1 Presenilin-1 CTF12 2.94 8.83PTGER2 Prostaglandin E2 receptor EP2 subtype 6.33 6.74 PTGER4Prostaglandin E2 receptor EP4 subtype 8.62 5.12 PTPRK Receptor-typetyrosine-protein phosphatase kappa 2.14 10.9 RET Extracellularcell-membrane anchored RET cadherin 120 2.38 12.3 kDa fragment SERINC3Serine incorporator 3 7.93 12.01 SIT1 Sodium- and chloride-dependenttransporter XTRP3 5.92 4.82 SLAMF1 Signaling lymphocytic activationmolecule 4.4 9.03 SLC29A1 Equilibrative nucleoside transporter 1 2.076.12 SLC39A6 Zinc transporter ZIP6 6.69 15.23 SLC7A5 Large neutral aminoacids transporter small subunit 1 3.79 10.98 STX4 Syntaxin-4 5.68 7.67TGFBR3 Transforming growth factor beta receptor type 3 7.55 7.29 TGOLN2Trans-Golgi network integral membrane protein 2 9.59 11.3 TLR1 Toll-likereceptor 1 2.34 4.57 TMED10 Transmembrane emp24 domain-containingprotein 10 9.34 12.24 TMEM97 Transmembrane protein 97 2.75 9.02 TNFTumor necrosis factor, soluble form 1.63 3.18 TNFRSF17 Tumor necrosisfactor receptor superfamily member 17 1.89 10.47 TNFRSF1B Tumor necrosisfactor-binding protein 2 5.51 9.4 VDAC1 Voltage-dependentanion-selective channel protein 1 6.52 11.5

TABLE 8 antigen markers expressed on the surface of both kidney tumorcells and T-cells Relative Relative Expression expression in colonAntigen Protein Name in T-Cell cancer cells ADORA3 Adenosine receptor A31.89 4.56 ATP11A Probable phospholipid-transporting ATPase IH 3.62 8.8BSG Basigin 4.77 11.34 BTN3A2 Butyrophilin subfamily 3 member A2 10.868.19 C10orf72 V-set and transmembrane domain-containing protein 4 2.046.85 CADM3 Cell adhesion molecule 3 3.57 6.39 CD8A T-cell surfaceglycoprotein CD8 alpha chain 10.35 6.6 CDH16 Cadherin-16 2.17 7.09 CDH4Cadherin-4 2.15 3.6 CDH5 Cadherin-5 2.5 9.55 CHL1 Processed neural celladhesion molecule L1-like protein 2.69 10.43 CHRNB1 Acetylcholinereceptor subunit beta 2.12 3.6 CLIC4 Chloride intracellular channelprotein 4 3.34 13.12 CNR1 Cannabinoid receptor 1 2.26 5.64 CRIM1Processed cysteine-rich motor neuron 1 protein 3.57 12.39 CSPG4Chondroitin sulfate proteoglycan 4 3.33 6.59 CYBB Cytochrome b-245 heavychain 2.86 8.07 EDNRB Endothelin B receptor 3.04 8.97 FLT1 Vascularendothelial growth factor receptor 1 2.75 8.5 FZD1 Frizzled-1 2.72 7.59GJC2 Gap junction gamma-2 protein 2.09 2.94 GLRB Glycine receptorsubunit beta 2.51 7.15 GPER G-protein coupled estrogen receptor 1 2.348.64 GPM6A Neuronal membrane glycoprotein M6-a 2.95 6.88 GPR162 ProbableG-protein coupled receptor 162 2.75 2.81 GPR4 G-protein coupled receptor4 2.93 8.09 GRM8 Metabotropic glutamate receptor 8 3.43 8.25 HLA-DPB1HLA class II histocompatibility antigen, DP beta 1 chain 9.93 13.99 HTR65-hydroxytryptamine receptor 6 4.83 10.07 INSR Insulin receptor subunitbeta 3.44 8.95 ITM2B Bri23 peptide 11.16 12.19 KCNJ1 ATP-sensitiveinward rectifier potassium channel 1 2.5 4.17 KDR Vascular endothelialgrowth factor receptor 2 2.99 9.95 KL Klotho peptide 2.83 7.59 LAIR1Leukocyte-associated immunoglobulin-like receptor 1 5.64 4.25 MFAP3Microfibril-associated glycoprotein 3 3.7 7.3 MFAP3LMicrofibrillar-associated protein 3-like 3.44 8.7 MICA MHC class Ipolypeptide-related sequence A 4.07 2.01 NCAM1 Neural cell adhesionmolecule 1 2.45 7.31 NOTCH3 Notch 3 intracellular domain 3.21 12.41NOTCH4 Notch 4 intracellular domain 5.89 8.84 OLR1 Oxidized low-densitylipoprotein receptor 1, soluble form 2.84 8.41 P2RY14 P2Y purinoceptor14 2.63 4.63 PCDH17 Protocadherin-17 1.7 7.36 PDGFRB Platelet-derivedgrowth factor receptor beta 2.68 10.48 PECAM1 Platelet endothelial celladhesion molecule 7.7 10.85 PLXND1 Plexin-D1 5.02 11.68 PPAP2B Lipidphosphate phosphohydrolase 3 4.17 12.46 PTAFR Platelet-activating factorreceptor 3.01 4.81 PTGER3 Prostaglandin E2 receptor EP3 subtype 4.7610.26 PTH1R Parathyroid hormone/parathyroid hormone-related peptide 2.357.31 receptor RAMP3 Receptor activity-modifying protein 3 1.79 8.84 ROR2Tyrosine-protein kinase transmembrane receptor ROR2 3.2 5.98 S1PR1Sphingosine 1-phosphate receptor 1 5.17 6.51 SCARB1 Scavenger receptorclass B member 1 3.01 10.4 SLC13A3 Solute carrier family 13 member 33.32 7.89 SLC16A4 Monocarboxylate transporter 5 2.88 12.54 SLC17A3Sodium-dependent phosphate transport protein 4 1.58 11.55 SLC28A1Sodium/nucleoside cotransporter 1 4.76 6.3 SLC2A5 Solute carrier family2, facilitated glucose transporter 2.74 8.5 member 5 SLC39A14 Zinctransporter ZIP14 2.66 11.63 SLC6A13 Sodium- and chloride-dependent GABAtransporter 2 2.75 7.44 SLC7A8 Large neutral amino acids transportersmall subunit 2 5.03 10.46 SLCO2A1 Solute carrier organic aniontransporter family member 2A1 3.46 8.06 TBXA2R Thromboxane A2 receptor4.01 3.64 TGFBR2 TGF-beta receptor type-2 10.41 10.94 THSD7AThrombospondin type-1 domain-containing protein 7A 3.05 8 TIE1Tyrosine-protein kinase receptor Tie-1 2.04 4.41 TNFRSF1A Tumor necrosisfactor-binding protein 1 6.84 10.52 TNFSF12 Tumor necrosis factor ligandsuperfamily member 12, 4.35 4.1 secreted form VAMP5 Vesicle-associatedmembrane protein 5 3.49 6.18

TABLE 9 antigen markers expressed on the surface of both liver tumorcells and T-cells Relative Relative Expression expression in colonAntigen Protein Name in T-Cell cancer cells ABCB4 Multidrug resistanceprotein 3 2.02 3.7 ADAM10 Disintegrin and metalloproteinasedomain-containing protein 9.42 9.41 10 ATR Anthrax toxin receptor 1 6.989.9 BST2 Bone marrow stromal antigen 2 7.38 12.45 BTN3A3 Butyrophilinsubfamily 3 member A3 9.72 7.48 C9 Complement component C9b 2.41 10.52CHRND Acetylcholine receptor subunit delta 2.43 4.05 CLDN14 Claudin-142.79 2.4 EPOR Erythropoietin receptor 4.67 10.55 ERBB2 Receptortyrosine-protein kinase erbB-2 2.36 14.12 F2RL3 Proteinase-activatedreceptor 4 2.17 2.61 GJB1 Gap junction beta-1 protein 2.96 9.4 GPR126G-protein coupled receptor 126 2.23 11.32 IL1R1 Interleukin-1 receptortype 1, soluble form 2.88 12.57 ITGB1 Integrin beta-1 8.76 13.48NAALADL1 N-acetylated-alpha-linked acidic dipeptidase-like protein 3.031.46 OR7A5 Olfactory receptor 7A5 1.51 3.83 SGCD Delta-sarcoglycan 3.997.21 SIGLEC6 Sialic acid-binding Ig-like lectin 6 3.57 3.49 SLC38A3Sodium-coupled neutral amino acid transporter 3 1.89 8.91 TFR2Transferrin receptor protein 2 2.74 10.47

TABLE 10 antigen markers expressed on the surface of both lung tumorcells and T-cells Relative Relative Expression expression in colonAntigen Protein Name in T-Cell cancer cells ABCB6 ATP-binding cassettesub-family B member 6, 2.88 9.82 mitochondrial ABCC1 Multidrugresistance-associated protein 1 7.05 8.16 ACCN1 Acid-sensing ion channel2 2.25 0.8 ADAM23 Disintegrin and metalloproteinase domain-containing2.51 4.73 protein 23 ADORA1 Adenosine receptor A1 4.49 8.22 ADORA2BAdenosine receptor A2b 1.66 7.5 AJAP1 Adherens junction-associatedprotein 1 1.85 6.24 APLP1 C30 2.22 6.02 AQP3 Aquaporin-3 8.38 13.88ATP10D Probable phospholipid-transporting ATPase VD 2.43 7.4 ATP1A3Sodium/potassium-transporting ATPase subunit alpha-3 3.01 3.13 ATP1B2Sodium/potassium-transporting ATPase subunit beta-2 3.21 3.8 ATP1B3Sodium/potassium-transporting ATPase subunit beta-3 8.6 14.26 AXLTyrosine-protein kinase receptor UFO 2.51 9.58 BEST1 Bestrophin-1 2.494.44 BTC Betacellulin 2.86 4.59 BTN3A1 Butyrophilin subfamily 3 memberA1 10.66 11.63 CALCR Calcitonin receptor 2.95 8.62 CALCRL Calcitoningene-related peptide type 1 receptor 2.12 7.67 CCR1 C-C chemokinereceptor type 1 2.63 9.77 CD163 Soluble CD163 2.66 8.76 CD300ACMRF35-like molecule 8 7.96 4.23 CD300A CMRF35-like molecule 8 2.29 4.23CD68 Macrosialin 4.02 8.92 CD74 HLA class II histocompatibility antigengamma chain 9.1 13.44 CD86 T-lymphocyte activation antigen CD86 2.935.04 CHRNA3 Neuronal acetylcholine receptor subunit alpha-3 2.54 4.62CHRNA3 Neuronal acetylcholine receptor subunit alpha-3 2 4.62 CKAP4Cytoskeleton-associated protein 4 6.15 11.94 CLCA2 Calcium-activatedchloride channel regulator 2, 35 kDa 2.99 9.81 form CLDN5 Claudin-5 3.667.73 CLSTN1 CTF1-alpha 8.26 12.51 CNIH3 Protein cornichon homolog 3 2.76.09 COMT Catechol O-methyltransferase 7.78 12.13 CSPG5 Chondroitinsulfate proteoglycan 5 2.84 5.69 CXCR6 C-X-C chemokine receptor type 63.16 3.91 CXCR7 Atypical chemokine receptor 3 2.5 8.95 DCHS1Protocadherin-16 4.29 2.28 DSC3 Desmocollin-2 2.82 8.95 DSG3Desmoglein-3 2.23 10.73 EGFR Epidermal growth factor receptor 3.8 10.92FAT2 Protocadherin Fat 2 2.25 9.29 FCER1G High affinity immunoglobulinepsilon receptor subunit 3.13 8.96 gamma FCGR1A High affinityimmunoglobulin gamma Fc receptor I 2.09 9.65 FLT4 Vascular endothelialgrowth factor receptor 3 3.19 3.36 FPR2 N-formyl peptide receptor 2 2.97.14 FURIN Furin 6.42 7.5 FZD6 Frizzled-6 2.64 10.45 GABBR2Gamma-aminobutyric acid type B receptor subunit 2 3.79 9.19 GABRB3Gamma-aminobutyric acid receptor subunit beta-3 2.46 8.83 GABRDGamma-aminobutyric acid receptor subunit delta 1.72 1.67 GABREGamma-aminobutyric acid receptor subunit epsilon 1.85 9.18 GIPR Gastricinhibitory polypeptide receptor 3.43 5.37 GJA1 Gap junction alpha-1protein 2.84 12.65 GJB3 Gap junction beta-3 protein 3.72 3.79 GJB5 Gapjunction beta-5 protein 1.77 6.69 GLRA2 Glycine receptor subunit alpha-22.26 6.15 GPR109B Hydroxycarboxylic acid receptor 3 1.77 2.91 GPR12G-protein coupled receptor 12 2 1.76 GPR176 Probable G-protein coupledreceptor 176 2.05 3.86 GPR50 Melatonin-related receptor 2.26 3.16 GRIK1Glutamate receptor ionotropic, kainate 1 4.66 5.65 GRIN2D Glutamatereceptor ionotropic, NMDA 2D 2.17 2.32 HCRTR1 Orexin receptor type 12.34 3.56 HLA-DPA1 HLA class II histocompatibility antigen, DP alpha 1chain 8.31 12.86 HLA-DQA1 HLA class II histocompatibility antigen, DQalpha 1 chain 2.35 11.44 HLA-DQB1 HLA class II histocompatibilityantigen, DQ beta 1 chain 7.4 12.71 HLA-DRA HLA class IIhistocompatibility antigen, DR alpha chain 6.42 14.18 HLA-DRB4 HLA classII histocompatibility antigen, DR beta 4 chain 2.72 11.24 IGSF9B Proteinturtle homolog B 3.92 2.81 IL1RAP Interleukin-1 receptor accessoryprotein 3.99 11.4 IL1RL1 Interleukin-1 receptor-like 1 2.55 5.15 IL4RSoluble interleukin-4 receptor subunit alpha 4.15 9.56 IL7RInterleukin-7 receptor subunit alpha 11.62 11.26 ITGA6 Integrin alpha-6light chain 7.99 12.76 JPH3 Junctophilin-3 2.34 2.5 KCNS3 Potassiumvoltage-gated channel subfamily S member 3 2.45 8.91 KIT Mast/stem cellgrowth factor receptor Kit 2.85 8.67 KITLG Soluble KIT ligand 2.58 7.27LILRB3 Leukocyte immunoglobulin-like receptor subfamily B 5.65 8.03member 3 LILRB4 Leukocyte immunoglobulin-like receptor subfamily B 3.1210.44 member 4 LPAR1 Lysophosphatidic acid receptor 1 4.12 5.47 LPHN3Latrophilin-3 2.85 6.43 MMP24 Processed matrix metalloproteinase-24 5.195.73 MPZ Myelin protein P0 2.56 3.63 MUC4 Mucin-4 beta chain 3.04 10.34NCKAP1L Nek-associated protein 1-like 6.69 7.51 NKG7 Protein NKG7 10.923.66 NOTCH2 Notch 2 intracellular domain 6.62 6.22 NRCAM Neuronal celladhesion molecule 2.78 8.16 NRG2 Neuregulin-2 3.55 9.22 NRXN1 Neurexin-12.56 5.33 NTRK2 BDNF/NT-3 growth factors receptor 2.56 10.7 NTSR1Neurotensin receptor type 1 1.74 9.74 P2RY1 P2Y purinoceptor 1 2.34 7.62P2RY6 P2Y purinoceptor 6 4.27 5.79 PCDH8 Protocadherin-8 2.67 9.29PCDHA3 Protocadherin alpha-3 2.14 3.54 PIK3IP1Phosphoinositide-3-kinase-interacting protein 1 8.68 3.47 PLXNA2Plexin-A2 2.88 7.3 PRR4 Processed poliovirus receptor-related protein 43.24 8.02 PTPRE Receptor-type tyrosine-protein phosphatase epsilon 6.037.92 PTPRO Receptor-type tyrosine-protein phosphatase U 10.46 9.01 PTPRUReceptor-type tyrosine-protein phosphatase U 3.72 6.18 RABAC1 PrenylatedRab acceptor protein 1 7.54 8.82 SCTR Secretin receptor 2.2 2.48 SECTM1Secreted and transmembrane protein 1 2.42 6.9 SGCE Epsilon-sarcoglycan2.15 9.65 SGCG Gamma-sarcoglycan 2.56 5.74 SLC16A3 Monocarboxylatetransporter 4 5.89 12.72 SLC16A7 Monocarboxylate transporter 2 5.39 6.97SLC20A2 Sodium-dependent phosphate transporter 2 2.51 12.69 SLC26A4Pendrin 3.57 9.39 SLC2A1 Solute carrier family 2, facilitated glucosetransporter 5.1 5.83 member 1 SLC4A7 Sodium bicarbonate cotransporter 34.89 8.7 SLCO3A1 Solute carrier organic anion transporter family member3A1 4.87 7.91 SYNE2 Nesprin-2 9.43 10.43 TACR2 Substance-K receptor 2.236.68 TFRC Transferrin receptor protein 1, serum form 7.32 14.31 TMEFF1Tomoregulin-1 3.22 5.05 TMPRSS11D Transmembrane protease serine 11Dcatalytic chain 2.35 8.32

TABLE 11 antigen markers expressed on the surface of both ovary tumorcells and T-cells; Relative Relative Expression expression in colonAntigen Protein Name in T-Cell cancer cells ACVR2B Activin receptortype-2B 2.1 4.26 ADAM28 Disintegrin and metalloproteinasedomain-containing 2.83 9.22 protein 28 ADRA2C Alpha-2C adrenergicreceptor 4.6 5.13 ATP2B1 Plasma membrane calcium-transporting ATPase 15.3 11.49 ATP2B4 Plasma membrane calcium-transporting ATPase 4 8.21 10.1ATP7A Copper-transporting ATPase 1 3.91 7.31 CD200 OX-2 membraneglycoprotein 2.83 10.51 CD47 Leukocyte surface antigen CD47 9.88 10.42CDH12 Cadherin-12 2.31 5.91 CDH18 Cadherin-18 2.28 4.79 CDH2 Cadherin-23.72 11.97 CDH6 Cadherin-6 2.77 8.68 CDIPT CDP-diacylglycerol--inositol3-phosphatidyltransferase 8.88 10.73 CELSR2 Cadherin EGF LAG seven-passG-type receptor 2 2.66 8.38 CHRNA1 Acetylcholine receptor subunit alpha2.42 5.71 CLSTN3 Calsyntenin-3 3.87 4.54 CX3CR1 CX3C chemokine receptor1 9 11.42 DDR1 Epithelial discoidin domain-containing receptor 1 3.8312.36 EPHA1 Ephrin type-A receptor 1 2.02 5.96 EPHA4 Ephrin type-Areceptor 4 2.39 8.56 ERBB4 ERBB4 intracellular domain 2.29 9.76 FGFR1Fibroblast growth factor receptor 1 5.42 11.4 FGFR3 Fibroblast growthfactor receptor 3 2.95 11.35 FZD2 Frizzled-2 1.91 8.06 FZD7 Frizzled-72.55 10.24 GJA4 Gap junction alpha-4 protein 2.04 6.7 GPR125 ProbableG-protein coupled receptor 125 2.35 7.88 GPR56 GPR56 C-terminal fragment8.6 11.27 GPR64 G-protein coupled receptor 64 2.04 8.57 GPRC5B G-proteincoupled receptor family C group 5 member B 1.96 10.29 GRIA2 Glutamatereceptor 2 1.96 11.78 GRIK5 Glutamate receptor ionotropic, kainate 55.79 3.36 GRIN2A Glutamate receptor ionotropic, NMDA 2A 1.68 2.96 HEG1Protein HEG homolog 1 4.8 10.1 HRH1 Histamine H1 receptor 2.31 6.26HTR3A 5-hydroxytryptamine receptor 3A 2.1 9.35 IFITM2 Interferon-inducedtransmembrane protein 2 10.27 11.36 IFITM3 Interferon-inducedtransmembrane protein 3 8.55 13.48 KCNH2 Potassium voltage-gated channelsubfamily H member 2 2.09 5.36 KCNJ12 ATP-sensitive inward rectifierpotassium channel 12 2.29 2.21 L1CAM Neural cell adhesion molecule L12.61 8.73 LGR5 Leucine-rich repeat-containing 2.45 12.12 G-proteincoupled receptor 5 LPHN1 Latrophilin-1 4.5 5.56 LPHN1 Latrophilin-1 1.635.56 LPHN2 Latrophilin-2 1.93 7.14 MGA Glucoamylase 5.15 5.65 NEO1Neogenin 1.85 10.31 NPTN Neuroplastin 8.46 13.14 NRG1 Neuregulin-1 2.616.53 NTRK1 High affinity nerve growth factor receptor 2.09 2.49 PCDH7Protocadherin-7 2.89 8.52 PCDH9 Protocadherin-9 2.99 6.15 PDGFRAPlatelet-derived growth factor receptor alpha 3.69 8.44 PDGFRAPlatelet-derived growth factor receptor alpha 2.26 8.44 PLXNB1 Plexin-B12.26 6.71 PLXNB2 Plexin-B2 3.1 10.68 PODXL Podocalyxin 2.73 11.41 PRSS8Prostasin heavy chain 2.07 10.77 PTH2R Parathyroid hormone 2 receptor1.85 8.67 PVRL3 Poliovirus receptor-related protein 3 2.56 10.15 SCNN1AAmiloride-sensitive sodium channel subunit alpha 5.97 10.63 SLC29A2Equilibrative nucleoside transporter 2 2.93 1.89 SSPN Sarcospan 3.499.16 STAR Heat-stable enterotoxin receptor 2.36 7.13 TGFA Transforminggrowth factor alpha 2.64 1.71 TMED1 Transmembrane emp24domain-containing protein 1 4.79 9.3 TMEM59 Transmembrane protein 598.83 12.74 TNFRSF25 Tumor necrosis factor receptor superfamily member 257.53 4.27 TYRO3 Tyrosine-protein kinase receptor TYRO3 4.11 10.27 UPK2Uroplakin-2 2.29 7.49

TABLE 12 antigen markers expressed on the surface of both pancreas tumorcells and T-cells Relative Relative Expression expression in colonAntigen Protein Name in T-Cell cancer cells ADAM9 Disintegrin andmetalloproteinase domain-containing 3.49 10.99 protein 9 B4GALT1Processed beta-1,4-galactosyltransferase 1 7.44 8.99 BDKRB2 B2bradykinin receptor 2.52 4.44 CA9 Carbonic anhydrase 9 3.34 11.9 CACNA1CVoltage-dependent L-type calcium channel subunit alpha- 2.36 4.54 1CCD58 Lymphocyte function-associated antigen 3 6.51 8.16 CDH11Cadherin-11 2.85 10.38 CDH3 Cadherin-3 1.96 10.91 CFTR Cystic fibrosistransmembrane conductance regulator 3.12 11.45 CHRNB4 Neuronalacetylcholine receptor subunit beta-4 2.38 0.66 CLDN10 Claudin-10 2.3611.5 CXCR4 C-X-C chemokine receptor type 4 11.74 10.98 DAG1Beta-dystroglycan 5.65 10.98 DDR2 Discoidin domain-containing receptor 22.34 8 DMPK Myotonin-protein kinase 3.7 4.21 FAT1 Protocadherin Fat 1,nuclear form 3.3 12.45 HTR2B 5-hydroxytryptamine receptor 2B 2.22 7.73LDLR Low-density lipoprotein receptor 2.93 12.14 NCKAP1 Nek-associatedprotein 1 3.34 11.99 PMP22 Peripheral myelin protein 22 2.09 10.66PNPLA2 Patatin-like phospholipase domain-containing protein 2 5.46 3.45PNPLA2 Patatin-like phospholipase domain-containing protein 2 2.35 3.45TEK Angiopoietin-1 receptor 3.87 8.52 TGFBR1 TGF-beta receptor type-12.17 4.3

TABLE 13 antigen markers expressed on the surface of both prostate tumorcells and T-cells Relative Relative Expression expression in colonAntigen Protein Name in T-Cell cancer cells ACCN3 Acid-sensing ionchannel 3 2.47 2.03 ADRB1 Beta-1 adrenergic receptor 2.85 5.09 ADRB2Beta-2 adrenergic receptor 5.74 9.43 AGTR1 Type-1 angiotensin IIreceptor 2.81 11.62 APLP2 Amyloid-like protein 2 7.06 13.06 ATP1A2Sodium/potassium-transporting ATPase subunit alpha-2 3.07 7.55 ATP8A1Probable phospholipid-transporting ATPase IA 7.23 9.16 CADM1 Celladhesion molecule 1 4.42 12.28 CHRM3 Muscarinic acetylcholine receptorM3 1.85 9.23 CHRNA2 Neuronal acetylcholine receptor subunit alpha-2 2.835.34 CXADR Coxsackievirus and adenovirus receptor 3.31 12.74 DPP4Dipeptidyl peptidase 4 soluble form 6.42 11.22 ECE1Endothelin-converting enzyme 1 7.14 4.7 ENPP4Bis(5′-adenosyl)-triphosphatase ENPP4 6.57 7.49 EPHA3 Ephrin type-Areceptor 3 2.84 7.85 ERG Potassium voltage-gated channel subfamily Hmember 2 2.72 11.3 FAM38A Piezo-type mechanosensitive ion channelcomponent 1 8.4 9.57 FOLH1 Glutamate carboxypeptidase 2 2.96 13.18GABRA2 Gamma-aminobutyric acid receptor subunit alpha-2 3 6.42 GHRGrowth hormone-binding protein 2.52 6.84 GPM6B Neuronal membraneglycoprotein M6-b 3.22 6.56 GPR116 Probable G-protein coupled receptor116 3.69 10.09 HBEGF Heparin-binding EGF-like growth factor 2.87 8.12JAM3 Junctional adhesion molecule C 4.29 7.26 KCND3 Potassiumvoltage-gated channel subfamily D member 3 3.09 9.77 LIFR Leukemiainhibitory factor receptor 2.71 6.8 LRBA Lipopolysaccharide-responsiveand beige-like anchor 5.35 9.26 protein MME Neprilysin 2.62 8.05 NOVPlexin-A1 2.43 10.41 NRP1 Neuropilin-1 3.17 7.85 OPRK1 Kappa-type opioidreceptor 2.07 4.92 PLXNB3 Plexin-B3 2.57 3.59 PPAP2A Lipid phosphatephosphohydrolase 1 3.6 11.55 SCAMP5 Secretory carrier-associatedmembrane protein 5 3.03 8.43 SLC23A2 Solute carrier family 23 member 23.55 7.04 SLC2A4 Solute carrier family 2, facilitated glucosetransporter 2.67 5.96 member 4 SLC36A1 Proton-coupled amino acidtransporter 1 3.38 9.28 SLC4A4 Electrogenic sodium bicarbonatecotransporter 1 3.14 11.29 STIM1 Stromal interaction molecule 1 3.686.51 TMPRSS2 Transmembrane protease serine 2 catalytic chain 2.67 9.63TRPV6 Transient receptor potential cation channel subfamily V 4.84 8.09member 6 VIPR1 Vasoactive intestinal polypeptide receptor 1 4.41 7.73YIPF3 Protein YIPF3, 36 kDa form III 4 4.3

TABLE 14 antigen markers expressed on the surface of T-cells andoverexpressed in liquid tumor cells (ALL, AML, CML, MDS, CLL, CTRL)Relative expression Antigen Protein Name on T cell CD63 CD63 antigen0.83 CXCR4 C-X-C chemokine receptor type 4 0.82 IFITM2Interferon-induced transmembrane protein 2 0.82 ITM2B Bri23 peptide 0.81BTF3 Butyrophilin subfamily 3 member A2 0.8 HLA-DRB1 HLA class IIhistocompatibility antigen, DRB1-12 beta chain 0.79 HLA-DRA HLA class IIhistocompatibility antigen, DR alpha chain 0.78 IFITM3Interferon-induced transmembrane protein 3 0.78 NKG7 Protein NKG7 0.78FCER1G High affinity immunoglobulin epsilon receptor subunit gamma 0.78IFITM1 Interferon-induced transmembrane protein 1 0.76 NPTN Neuroplastin0.76 GYPC Glycophorin-C 0.76 GPR160 Probable G-protein coupled receptor160 0.76 HLA-DPB1 HLA class II histocompatibility antigen, DP beta 1chain 0.75 BRI3 CT-BRI3 0.75 SLC38A2 Sodium-coupled neutral amino acidtransporter 2 0.74 C5AR1 C5a anaphylatoxin chemotactic receptor 1 0.74CDIPT CDP-diacylglycerol--inositol 3-phosphatidyltransferase 0.73TNFSF13B Tumor necrosis factor ligand superfamily member 13b, solubleform 0.73 CSF3R Granulocyte colony-stimulating factor receptor 0.73HLA-DPA1 HLA class II histocompatibility antigen, DP alpha 1 chain 0.71CD164 Sialomucin core protein 24 0.71 CD97 CD97 antigen subunit beta 0.7C3AR1 C3a anaphylatoxin chemotactic receptor 0.69 P2RY8 P2Y purinoceptor8 0.68 BSG Basigin 0.68 APLP2 Amyloid-like protein 2 0.67 TFRCTransferrin receptor protein 1, serum form 0.67 MGAM Glucoamylase 0.67GYPA Glycophorin-A 0.67 TMED10 Transmembrane emp24 domain-containingprotein 10 0.66 FCGRT IgG receptor FcRn large subunit p51 0.66 CKAP4Cytoskeleton-associated protein 4 0.66 DYSF Dysferlin 0.66 SPPL2A Signalpeptide peptidase-like 2A 0.65 LAMP2 Lysosome-associated membraneglycoprotein 2 0.65 SLC7A5 Large neutral amino acids transporter smallsubunit 1 0.65 TNFRSF1B Tumor necrosis factor-binding protein 2 0.64TREM1 Triggering receptor expressed on myeloid cells 1 0.64 GPR183G-protein coupled receptor 183 0.63 SERINC3 Serine incorporator 3 0.63CD58 Lymphocyte function-associated antigen 3 0.63 GYPB Glycophorin-B0.63 RABAC1 Prenylated Rab acceptor protein 1 0.62 KCNH2 Potassiumvoltage-gated channel subfamily H member 2 0.62 FPR1 fMet-Leu-Phereceptor 0.62 P2RY13 P2Y purinoceptor 13 0.62 CLEC5A C-type lectindomain family 5 member A 0.62 SLC7A7 Y + L amino acid transporter 1 0.61MICB MHC class I polypeptide-related sequence B 0.61 CD300LF CMRF35-likemolecule 1 0.61 GJB6 Gap junction beta-6 protein 0.61 ATP1A1Sodium/potassium-transporting ATPase subunit alpha-1 0.6 PTGER4Prostaglandin E2 receptor EP4 subtype 0.6 CD8A T-cell surfaceglycoprotein CD8 alpha chain 0.6 PTGER2 Prostaglandin E2 receptor EP2subtype 0.6 GPR97 Probable G-protein coupled receptor 97 0.6 IMP3 Signalpeptide peptidase-like 2A 0.59 LAMP1 Lysosome-associated membraneglycoprotein 1 0.59 LILRB3 Leukocyte immunoglobulin-like receptorsubfamily B member 3 0.59 GPR109B Hydroxycarboxylic acid receptor 3 0.59SAT2 Sodium-coupled neutral amino acid transporter 2 0.58 GPR65Psychosine receptor 0.58 AMICA1 Junctional adhesion molecule-like 0.58PAG1 Phosphoprotein associated with glycosphingolipid-enriched 0.58microdomains 1 ENPP4 Bis(5′-adenosyl)-triphosphatase ENPP4 0.57 SLC40A1Solute carrier family 40 member 1 0.57 OLR1 Oxidized low-densitylipoprotein receptor 1, soluble form 0.57 LRRC33 Negative regulator ofreactive oxygen species 0.56 IL7R Interleukin-7 receptor subunit alpha0.56 LAIR1 Leukocyte-associated immunoglobulin-like receptor 1 0.56ITM2C CT-BRI3 0.56 GPR84 G-protein coupled receptor 84 0.56 SLC12A7Solute carrier family 12 member 7 0.55 PTAFR Platelet-activating factorreceptor 0.55 CD33 Myeloid cell surface antigen CD33 0.55 SLC22A16Solute carrier family 22 member 16 0.55 CCR7 C-C chemokine receptor type7 0.54 TLR1 Toll-like receptor 1 0.54 TGOLN2 Trans-Golgi networkintegral membrane protein 2 0.54 YIPF3 Protein YIPF3, 36 kDa form III0.54 BST2 Bone marrow stromal antigen 2 0.54 MAGT1 Magnesium transporterprotein 1 0.54 TMEM173 Stimulator of interferon genes protein 0.54 ERMAPErythroid membrane-associated protein 0.54 CEACAM1 Carcinoembryonicantigen-related cell adhesion molecule 1 0.54 NIPA2 Magnesiumtransporter NIPA2 0.53 PECAM1 Platelet endothelial cell adhesionmolecule 0.53 CD1D Antigen-presenting glycoprotein CD1d 0.53 TMEM59Transmembrane protein 59 0.53 NCKAP1L Nek-associated protein 1-like 0.53FAS Tumor necrosis factor receptor superfamily member 6 0.53 IL6RInterleukin-6 receptor subunit alpha 0.53 TNFRSF1A Tumor necrosisfactor-binding protein 1 0.53 KEL Kell blood group glycoprotein 0.53TMEM149 IGF-like family receptor 1 0.52 SLC3A2 4F2 cell-surface antigenheavy chain 0.52 ORAI1 Calcium release-activated calcium channel protein1 0.52 XKR8 XK-related protein 8, processed form 0.52 C9orf46Plasminogen receptor (KT) 0.52 TMEM127 Transmembrane protein 127 0.52SLC2A1 Solute carrier family 2, facilitated glucose transporter member 10.52 FCGR1B High affinity immunoglobulin gamma Fc receptor IB 0.52 CXCR2C-X-C chemokine receptor type 2 0.52 IL4R Soluble interleukin-4 receptorsubunit alpha 0.51 HSD17B7 3-keto-steroid reductase 0.51 SEMA4DSemaphorin-4D 0.51 ZDHHC5 Palmitoyltransferase ZDHHC5 0.51 ADRB2 Beta-2adrenergic receptor 0.51 S1PR4 Sphingosine 1-phosphate receptor 4 0.51PILRA Paired immunoglobulin-like type 2 receptor alpha 0.51 LTB4RLeukotriene B4 receptor 1 0.51 SORT1 Sortilin 0.51 SLCO4C1 Solutecarrier organic anion transporter family member 4C1 0.51 ANO10Anoctamin-10 0.51 CLSTN1 CTF1-alpha 0.5 RHBDF2 Inactive rhomboid protein2 0.5 CCR1 C-C chemokine receptor type 1 0.5 EPCAM Epithelial celladhesion molecule 0.5 PNPLA2 Patatin-like phospholipasedomain-containing protein 2 0.49 SLC12A6 Solute carrier family 12 member6 0.49 SLC30A1 Zinc transporter 1 0.49 GPR27 Probable G-protein coupledreceptor 27 0.49 EPOR Erythropoietin receptor 0.49 CD79A B-cell antigenreceptor complex-associated protein alpha chain 0.48 HLA-DQB1 HLA classII histocompatibility antigen, DQ beta 1 chain 0.48 HBP1Glycosylphosphatidylinositol-anchored high density lipoprotein- 0.48binding protein 1 ABCA7 ATP-binding cassette sub-family A member 7 0.48RAG1AP1 Sugar transporter SWEET1 0.48 CD47 Leukocyte surface antigenCD47 0.48 CXCL16 C-X-C motif chemokine 16 0.48 SLC14A1 Urea transporter1 0.48 TGFBR2 TGF-beta receptor type-2 0.47 LRBALipopolysaccharide-responsive and beige-like anchor protein 0.47 MFSD5Molybdate-anion transporter 0.47 RELT Tumor necrosis factor receptorsuperfamily member 19L 0.47 ATP2B4 Plasma membrane calcium-transportingATPase 4 0.47 FURIN Furin 0.47 GAPT Protein GAPT 0.47 NFAM1 NFATactivation molecule 1 0.47 ATP2B1 Plasma membrane calcium-transportingATPase 1 0.46 SLC26A11 Sodium-independent sulfate anion transporter 0.46STX4 Syntaxin-4 0.46 NAT1 Sodium-coupled neutral amino acid transporter3 0.46 STIM1 Stromal interaction molecule 1 0.46 SLC39A4 Zinctransporter ZIP4 0.46 ESYT2 Extended synaptotagmin-2 0.46 TM7SF3Transmembrane 7 superfamily member 3 0.46 SEMA4A Semaphorin-4A 0.46 CYBBCytochrome b-245 heavy chain 0.46 FCAR Immunoglobulin alpha Fc receptor0.46 GABBR1 Gamma-aminobutyric acid type B receptor subunit 1 0.45 CD53Leukocyte surface antigen CD53 0.45 SIGLEC10 Sialic acid-binding Ig-likelectin 10 0.45 S1PR1 Sphingosine 1-phosphate receptor 1 0.45 BTN3A2Butyrophilin subfamily 3 member A2 0.45 NOTCH2 Notch 2 intracellulardomain 0.45 PIK3IP1 Phosphoinositide-3-kinase-interacting protein 1 0.45FAM168B Myelin-associated neurite-outgrowth inhibitor 0.45 LPAR2Lysophosphatidic acid receptor 2 0.45 ATP1B3Sodium/potassium-transporting ATPase subunit beta-3 0.45 FLVCR1 Felineleukemia virus subgroup C receptor-related protein 1 0.45 SECTM1Secreted and transmembrane protein 1 0.45 SLC38A5 Sodium-coupled neutralamino acid transporter 5 0.45 GPR18 N-arachidonyl glycine receptor 0.44LMBR1L Protein LMBR1L 0.44 ABCC1 Multidrug resistance-associated protein1 0.44 SLC22A18 Solute carrier family 22 member 18 0.44 CSF1R Macrophagecolony-stimulating factor 1 receptor 0.44 EMR1 EGF-likemodule-containing mucin-like hormone receptor-like 1 0.44 FPR2 N-formylpeptide receptor 2 0.44 KIT Mast/stem cell growth factor receptor Kit0.44 MS4A1 B-lymphocyte antigen CD20 0.43 MICA MHC class Ipolypeptide-related sequence A 0.43 GPR172A Solute carrier family 52,riboflavin transporter, member 2 0.43 F11R Junctional adhesion moleculeA 0.43 ADAM10 Disintegrin and metalloproteinase domain-containingprotein 10 0.43 FAM38A Piezo-type mechanosensitive ion channel component1 0.43 CD68 Macrosialin 0.43 SLC26A6 Solute carrier family 26 member 60.43 MCOLN1 Mucolipin-1 0.43 SLCO3A1 Solute carrier organic aniontransporter family member 3A1 0.43 PPAP2B Lipid phosphatephosphohydrolase 3 0.43 ICAM4 Intercellular adhesion molecule 4 0.43CXCR1 C-X-C chemokine receptor type 1 0.43 CD300A CMRF35-like molecule 80.43 RELL1 RELT-like protein 1 0.43 TAPBPL Tapasin-related protein 0.42FCGR2C Low affinity immunoglobulin gamma Fc region receptor II-c 0.42SLC16A6 Monocarboxylate transporter 7 0.42 TMED1 Transmembrane emp24domain-containing protein 1 0.42 CD86 T-lymphocyte activation antigenCD86 0.42 SLC16A3 Monocarboxylate transporter 4 0.42 SLC2A5 Solutecarrier family 2, facilitated glucose transporter member 5 0.42 SLC29A1Equilibrative nucleoside transporter 1 0.42 SLC16A14 Monocarboxylatetransporter 14 0.42 P2RY2 P2Y purinoceptor 2 0.42 SUCNR1 Succinatereceptor 1 0.42 BTN3A1 Butyrophilin subfamily 3 member A1 0.41 LAT2Linker for activation of T-cells family member 2 0.41 PLXND1 Plexin-D10.41 ECE1 Endothelin-converting enzyme 1 0.41 TGFBR1 TGF-beta receptortype-1 0.41 CCRL2 C-C chemokine receptor-like 2 0.41 TFR2 Transferrinreceptor protein 2 0.41 SLC44A1 Choline transporter-like protein 1 0.41ITGA6 Integrin alpha-6 light chain 0.41 PMP22 Peripheral myelin protein22 0.41 LAX1 Lymphocyte transmembrane adapter 1 0.4 AMIGO2Amphoterin-induced protein 2 0.4 SLC38A1 Sodium-coupled neutral aminoacid transporter 1 0.4 SLC41A1 Solute carrier family 41 member 1 0.4C2orf89 Metalloprotease TIKI1 0.4 ABCC10 Multidrug resistance-associatedprotein 7 0.4 CLDN15 Claudin-15 0.4 SLC39A6 Zinc transporter ZIP6 0.4SLC16A5 Monocarboxylate transporter 6 0.4 TTYH3 Protein tweety homolog 30.4 ATP7A Copper-transporting ATPase 1 0.4 COMT CatecholO-methyltransferase 0.4 SLC17A5 Sialin 0.4 TMIGD2 Transmembrane andimmunoglobulin domain-containing protein 2 0.4 CLEC7A C-type lectindomain family 7 member A 0.4 SLC31A1 High affinity copper uptake protein1 0.4 LRRC4 Leucine-rich repeat-containing protein 4 0.4 P2RY10 PutativeP2Y purinoceptor 10 0.39 ATP10D Probable phospholipid-transportingATPase VD 0.39 BTN3A3 Butyrophilin subfamily 3 member A3 0.39 LIME1Lck-interacting transmembrane adapter 1 0.39 TNF Tumor necrosis factor,soluble form 0.39 PAQR8 Membrane progestin receptor beta 0.39 OXER1Oxoeicosanoid receptor 1 0.39 TRAT1 T-cell receptor-associatedtransmembrane adapter 1 0.39 GPBAR1 G-protein coupled bile acid receptor1 0.39 SLC36A1 Proton-coupled amino acid transporter 1 0.39 PTPREReceptor-type tyrosine-protein phosphatase epsilon 0.39 PROM1 Prominin-10.39 CD74 HLA class II histocompatibility antigen gamma chain 0.38 CNSTConsortin 0.38 TMEM49 Vacuole membrane protein 1 0.38 CLIC4 Chlorideintracellular channel protein 4 0.38 NAALADL1 N-acetylated-alpha-linkedacidic dipeptidase-like protein 0.38 ANTXR2 Anthrax toxin receptor 20.38 FGFR1 Fibroblast growth factor receptor 1 0.38 IL1RAP Interleukin-1receptor accessory protein 0.38 ATP1B2 Sodium/potassium-transportingATPase subunit beta-2 0.38 ABCG2 ATP-binding cassette sub-family Gmember 2 0.38 CLEC12A C-type lectin domain family 12 member A 0.38HLA-DQA1 HLA class II histocompatibility antigen, DQ alpha 1 chain 0.37B4GALT1 Processed beta-1,4-galactosyltransferase 1 0.37 CNNM3 Metaltransporter CNNM3 0.37 ATP1B1 Sodium/potassium-transporting ATPasesubunit beta-1 0.37 SLC39A1 Zinc transporter ZIP1 0.37 ATRN Attractin0.37 CYSLTR1 Cysteinyl leukotriene receptor 1 0.37 TRPV2 Transientreceptor potential cation channel subfamily V member 2 0.37 SLC27A1Long-chain fatty acid transport protein 1 0.37 GPR171 Probable G-proteincoupled receptor 171 0.37 DAGLB Sn1-specific diacylglycerol lipase beta0.37 KCNQ1 Potassium voltage-gated channel subfamily KQT member 1 0.37FZD6 Frizzled-6 0.37 CSF2RA Granulocyte-macrophage colony-stimulatingfactor receptor subunit 0.37 alpha PTH2R Parathyroid hormone 2 receptor0.37 MARCH1 E3 ubiquitin-protein ligase MARCH1 0.36 BACE2 Beta-secretase2 0.36 CD5 T-cell surface glycoprotein CD5 0.36 TMEM219 Insulin-likegrowth factor-binding protein 3 receptor 0.36 XPR1 Xenotropic andpolytropic retrovirus receptor 1 0.36 CD1C T-cell surface glycoproteinCD1c 0.36 CNNM2 Metal transporter CNNM2 0.36 TMEM88 Transmembraneprotein 88 0.36 ICOS Inducible T-cell costimulator 0.36 KLRG1 Killercell lectin-like receptor subfamily G member 1 0.36 LRP8 Low-densitylipoprotein receptor-related protein 8 0.36 F2R Proteinase-activatedreceptor 1 0.36 HM13 Minor histocompatibility antigen H13 0.36 EMR2EGF-like module-containing mucin-like hormone receptor-like 2 0.36TREML1 Trem-like transcript 1 protein 0.36 C17orf60 Allergin-1 0.36GPR146 Probable G-protein coupled receptor 146 0.36 SLAMF6 SLAM familymember 6 0.35 SLC7A6 Y + L amino acid transporter 2 0.35 RELL2 RELT-likeprotein 2 0.35 LGR6 Leucine-rich repeat-containing G-protein coupledreceptor 6 0.35 PANX1 Pannexin-1 0.35 C18orf1 Low-density lipoproteinreceptor class A domain-containing protein 0.35 4 SLMAP Sarcolemmalmembrane-associated protein 0.35 CCR5 C-C chemokine receptor type 5 0.35MUC1 Mucin-1 subunit beta 0.35 EMR3 EGF-like module-containingmucin-like hormone receptor-like 3 0.35 subunit beta COL23A1 Collagenalpha-1 (XXIII) chain 0.35 OR2W3 Olfactory receptor 2W3 0.35 LNPEPLeucyl-cystinyl aminopeptidase, pregnancy serum form 0.34 PRR7Proline-rich protein 7 0.34 NOTCH1 Notch 1 intracellular domain 0.34RFT1 Solute carrier family 52, riboflavin transporter, member 1 0.34TNFRSF25 Tumor necrosis factor receptor superfamily member 25 0.34 ANO6Anoctamin-6 0.34 AQP3 Aquaporin-3 0.34 ADAM9 Disintegrin andmetalloproteinase domain-containing protein 9 0.34 INSR Insulin receptorsubunit beta 0.34 FZD5 Frizzled-5 0.34 ERG Potassium voltage-gatedchannel subfamily H member 2 0.34 MME Neprilysin 0.34 FCGR2B Lowaffinity immunoglobulin gamma Fc region receptor II-b 0.33 LSRLipolysis-stimulated lipoprotein receptor 0.33 DDR1 Epithelial discoidindomain-containing receptor 1 0.33 CNR2 Cannabinoid receptor 2 0.33 ATRAnthrax toxin receptor 1 0.33 P2RY14 P2Y purinoceptor 14 0.33 VEZTVezatin 0.33 ALG10B Putative Dol-P-Glc: Glc(2)Man(9)GlcNAc(2)-PP-Dolalpha-1,2- 0.33 glucosyltransferase PAQR7 Membrane progestin receptoralpha 0.33 FLT3LG Fms-related tyrosine kinase 3 ligand 0.33 CD40LG CD40ligand, soluble form 0.33 FCGR2A Low affinity immunoglobulin gamma Fcregion receptor II-a 0.33 CLDN12 Claudin-12 0.33 GP6 Plateletglycoprotein VI 0.33 EPHB4 Ephrin type-B receptor 4 0.33 SEMA4CSemaphorin-4C 0.33 CD300C CMRF35-like molecule 6 0.33 PEAR1 Plateletendothelial aggregation receptor 1 0.33 FFAR2 Free fatty acid receptor 20.33 SLC2A6 Solute carrier family 2, facilitated glucose transportermember 6 0.32 TMEM150A Transmembrane protein 150A 0.32 ANO8 Anoctamin-80.32 CD200R1 Cell surface glycoprotein CD200 receptor 1 0.32 FCER1A Highaffinity immunoglobulin epsilon receptor subunit alpha 0.32 BEST1Bestrophin-1 0.32 CLDN5 Claudin-5 0.32 SLC47A1 Multidrug and toxinextrusion protein 1 0.32 SLC5A10 Sodium/glucose cotransporter 5 0.32CD40 Tumor necrosis factor receptor superfamily member 5 0.31 ANO9Anoctamin-9 0.31 CLEC2D C-type lectin domain family 2 member D 0.31VIPR1 Vasoactive intestinal polypeptide receptor 1 0.31 SLC16A7Monocarboxylate transporter 2 0.31 UTS2R Urotensin-2 receptor 0.31CLSTN3 Calsyntenin-3 0.31 GPR35 G-protein coupled receptor 35 0.31 SYT15Synaptotagmin-15 0.31 FAM57A Protein FAM57A 0.31 CD8B T-cell surfaceglycoprotein CD8 beta chain 0.31 IL17RC Interleukin-17 receptor C 0.31GLDN Gliomedin 0.31 FZD2 Frizzled-2 0.31 KCNA3 Potassium voltage-gatedchannel subfamily A member 3 0.3 MGA Glucoamylase 0.3 GPR1 G-proteincoupled receptor 1 0.3 IL6ST Interleukin-6 receptor subunit beta 0.3PCDHGB5 Protocadherin gamma-B5 0.3 OR1I1 Olfactory receptor 1I1 0.3PTH1R Parathyroid hormone/parathyroid hormone-related peptide receptor0.3 NLGN2 Neuroligin-2 0.3 MMP24 Processed matrix metalloproteinase-240.3 CDH22 Cadherin-22 0.3 TNFRSF8 Tumor necrosis factor receptorsuperfamily member 8 0.3 CHRNG Acetylcholine receptor subunit gamma 0.3PSEN1 Presenilin-1 CTF12 0.3 GPR114 Probable G-protein coupled receptor114 0.3 PLXNB2 Plexin-B2 0.3 CHRNA2 Neuronal acetylcholine receptorsubunit alpha-2 0.3 GPR34 Probable G-protein coupled receptor 34 0.3LPAR6 Lysophosphatidic acid receptor 6 0.3 ATP8A1 Probablephospholipid-transporting ATPase IA 0.3 FZD1 Frizzled-1 0.3 CCR2 C-Cchemokine receptor type 2 0.3 P2RY1 P2Y purinoceptor 1 0.3 SLC16A9Monocarboxylate transporter 9 0.3 C20orf103 Lysosome-associated membraneglycoprotein 5 0.3 ADORA2B Adenosine receptor A2b 0.3 CLEC12B C-typelectin domain family 12 member B 0.3 FCRL3 Fc receptor-like protein 30.29 CD180 CD180 antigen 0.29 TIGIT T-cell immunoreceptor with Ig andITIM domains 0.29 PPAP2A Lipid phosphate phosphohydrolase 1 0.29 ATP11CProbable phospholipid-transporting ATPase IG 0.29 TNFRSF17 Tumornecrosis factor receptor superfamily member 17 0.29 TNFSF12 Tumornecrosis factor ligand superfamily member 12, secreted form 0.29 TBXA2RThromboxane A2 receptor 0.29 OR3A3 Olfactory receptor 3A3 0.29 GPR153Probable G-protein coupled receptor 153 0.29 ATP11A Probablephospholipid-transporting ATPase IH 0.29 LRFN1 Leucine-rich repeat andfibronectin type III domain-containing 0.29 protein 1 OR51B2 Olfactoryreceptor 51B2 0.29 KCNS1 Potassium voltage-gated channel subfamily Smember 1 0.29 OR12D2 Olfactory receptor 12D2 0.29 GRM4 Metabotropicglutamate receptor 4 0.29 NEO1 Neogenin 0.29 DRD5 D(1B) dopaminereceptor 0.29 PLXDC1 Plexin domain-containing protein 1 0.29 GPR157Probable G-protein coupled receptor 157 0.29 CD300LB CMRF35-likemolecule 7 0.29 MARVELD1 MARVEL domain-containing protein 1 0.29 MFAP3Microfibril-associated glycoprotein 3 0.29 CHRNB1 Acetylcholine receptorsubunit beta 0.29 PVRL2 Poliovirus receptor-related protein 2 0.29 F2RL1Proteinase-activated receptor 2, alternate cleaved 2 0.29 GPR124G-protein coupled receptor 124 0.29 BACE1 Beta-secretase 1 0.29 C6orf105Androgen-dependent TFPI-regulating protein 0.28 CXCR3 C-X-C chemokinereceptor type 3 0.28 IGSF8 Immunoglobulin superfamily member 8 0.28ATP8B1 Probable phospholipid-transporting ATPase IC 0.28 TP53I13 Tumorprotein p53-inducible protein 13 0.28 MC1R Melanocyte-stimulatinghormone receptor 0.28 CD84 SLAM family member 5 0.28 CALHM1 Calciumhomeostasis modulator protein 1 0.28 CHRNA6 Neuronal acetylcholinereceptor subunit alpha-6 0.28 CDH10 Cadherin-10 0.28 SLC16A1Monocarboxylate transporter 1 0.28 GPRC5D G-protein coupled receptorfamily C group 5 member D 0.28 AGER Advanced glycosylation endproduct-specific receptor 0.28 FASLG FasL intracellular domain 0.28GPR56 GPR56 C-terminal fragment 0.28 SIGLEC1 Sialoadhesin 0.28 KIR2DL5AKiller cell immunoglobulin-like receptor 2DL5A 0.28 PLB1Lysophospholipase 0.28 CD200 OX-2 membrane glycoprotein 0.27 ADAM28Disintegrin and metalloproteinase domain-containing protein 28 0.27 SIT1Sodium- and chloride-dependent transporter XTRP3 0.27 SLC23A2 Solutecarrier family 23 member 2 0.27 CCR10 C-C chemokine receptor type 100.27 PRR4 Processed poliovirus receptor-related protein 4 0.27 GJD2 Gapjunction delta-2 protein 0.27 SLC2A8 Solute carrier family 2,facilitated glucose transporter member 8 0.27 CD209 CD209 antigen 0.27CD274 Programmed cell death 1 ligand 1 0.27 PROM2 Prominin-2 0.27ATP6V0A2 V-type proton ATPase 116 kDa subunit a isoform 2 0.27 MPZMyelin protein P0 0.27 TNFRSF18 Tumor necrosis factor receptorsuperfamily member 18 0.27 MFSD2A Major facilitator superfamilydomain-containing protein 2A 0.27 HEG1 Protein HEG homolog 1 0.27 OXTROxytocin receptor 0.27 CD99L2 CD99 antigen-like protein 2 0.27 LILRB4Leukocyte immunoglobulin-like receptor subfamily B member 4 0.27 SMAGPSmall cell adhesion glycoprotein 0.27 OR51I2 Olfactory receptor 51I20.27 LY6G6D Lymphocyte antigen 6 complex locus protein G6f 0.27 KCNQ4Potassium voltage-gated channel subfamily KQT member 4 0.27 HRH2Histamine H2 receptor 0.27 SLC39A2 Zinc transporter ZIP2 0.27 CLDN10Claudin-10 0.27 GPM6B Neuronal membrane glycoprotein M6-b 0.27 STEAP4Metalloreductase STEAP4 0.27 APOLD1 Apolipoprotein L domain-containingprotein 1 0.27 S1PR3 Sphingosine 1-phosphate receptor 3 0.27 SGMS2Phosphatidylcholine: ceramide cholinephosphotransferase 2 0.27 KIR2DS5Killer cell immunoglobulin-like receptor 2DS5 0.27 STAR Heat-stableenterotoxin receptor 0.27 NIPA1 Magnesium transporter NIPA1 0.26 CNNM4Metal transporter CNNM4 0.26 SLAMF1 Signaling lymphocytic activationmolecule 0.26 KIAA1919 Sodium-dependent glucose transporter 1 0.26 TLR6Toll-like receptor 6 0.26 CRB3 Protein crumbs homolog 3 0.26 SLC12A9Solute carrier family 12 member 9 0.26 GPR68 Ovarian cancer G-proteincoupled receptor 1 0.26 OR51J1 Olfactory receptor 51J1 0.26 TREML2Trem-like transcript 2 protein 0.26 GPR176 Probable G-protein coupledreceptor 176 0.26 FLVCR2 Feline leukemia virus subgroup Creceptor-related protein 2 0.26 LPAR1 Lysophosphatidic acid receptor 10.26 PANX2 Pannexin-2 0.26 SLC6A6 Sodium- and chloride-dependent taurinetransporter 0.26 PROKR2 Prokineticin receptor 2 0.26 CLDN9 Claudin-90.26 MYOF Myoferlin 0.26 LY6G6F Lymphocyte antigen 6 complex locusprotein G6f 0.26 ESAM Endothelial cell-selective adhesion molecule 0.26NCR3 Natural cytotoxicity triggering receptor 3 0.25 HLA-DQB2 HLA classII histocompatibility antigen, DQ beta 2 chain 0.25 SLC4A5 Electrogenicsodium bicarbonate cotransporter 4 0.25 P2RY4 P2Y purinoceptor 4 0.25ABCB1 Multidrug resistance protein 1 0.25 SLC9A1 Sodium/hydrogenexchanger 1 0.25 CELSR2 Cadherin EGF LAG seven-pass G-type receptor 20.25 SYT8 Synaptotagmin-8 0.25 PCDHA9 Protocadherin alpha-9 0.25 TMEM204Transmembrane protein 204 0.25 PTPRJ Receptor-type tyrosine-proteinphosphatase eta 0.25 GRPR Gastrin-releasing peptide receptor 0.25 SEMA6BSemaphorin-6B 0.25 CLCN5 H(+)/Cl(−) exchange transporter 5 0.25 GLRA2Glycine receptor subunit alpha-2 0.25 PLVAP Plasmalemmavesicle-associated protein 0.25 ACVR1B Activin receptor type-1B 0.25JAM3 Junctional adhesion molecule C 0.25 LDLRAD3 Low-density lipoproteinreceptor class A domain-containing protein 0.25 3 XG Glycoprotein Xg0.25 SLC2A11 Solute carrier family 2, facilitated glucose transportermember 11 0.24 PCDH9 Protocadherin-9 0.24 VAMP5 Vesicle-associatedmembrane protein 5 0.24 CDHR2 Cadherin-related family member 2 0.24 DRD2D(2) dopamine receptor 0.24 LRIG2 Leucine-rich repeats andimmunoglobulin-like domains protein 2 0.24 RAMP3 Receptoractivity-modifying protein 3 0.24 SLC39A14 Zinc transporter ZIP14 0.24STRA6 Stimulated by retinoic acid gene 6 protein homolog 0.24 ADRA2CAlpha-2C adrenergic receptor 0.24 CLDN19 Claudin-19 0.24 CX3CR1 CX3Cchemokine receptor 1 0.24 CD79B B-cell antigen receptorcomplex-associated protein beta chain 0.24 KIR2DL2 Killer cellimmunoglobulin-like receptor 2DL2 0.24 CXCR7 Atypical chemokine receptor3 0.24 OR5L2 Olfactory receptor 5L2 0.24 LRRC52 Leucine-richrepeat-containing protein 52 0.24 JPH1 Junctophilin-1 0.24 ADORA1Adenosine receptor A1 0.24 GPRC5C G-protein coupled receptor family Cgroup 5 member C 0.24 RET Extracellular cell-membrane anchored RETcadherin 120 kDa 0.24 fragment PVR Poliovirus receptor 0.24 ITGB3Integrin beta-3 0.24 PTGIR Prostacyclin receptor 0.24 LPHN1Latrophilin-1 0.24 OR10J1 Olfactory receptor 10J1 0.24 MFAP3LMicrofibrillar-associated protein 3-like 0.24 GPNMB Transmembraneglycoprotein NMB 0.24 CELSR3 Cadherin EGF LAG seven-pass G-type receptor3 0.23 CCR6 C-C chemokine receptor-like 2 0.23 DMPK Myotonin-proteinkinase 0.23 UPK3B Uroplakin-3b 0.23 OR1D2 Olfactory receptor 1D2 0.23OR7D2 Olfactory receptor 7D2 0.23 ITGB1 Integrin beta-1 0.23 HRH3Histamine H3 receptor 0.23 GRIN2C Glutamate receptor ionotropic, NMDA 2C0.23 KIR3DL1 Killer cell immunoglobulin-like receptor 3DL1 0.23 EPHB2Ephrin type-B receptor 2 0.23 OR2S2 Olfactory receptor 2S2 0.23 KIR2DL4Killer cell immunoglobulin-like receptor 2DL4 0.23 CNNM1 Metaltransporter CNNM1 0.23 MARVELD2 MARVEL domain-containing protein 2 0.23CXCR6 C-X-C chemokine receptor type 6 0.23 NOV Plexin-A1 0.23 ABCB6ATP-binding cassette sub-family B member 6, mitochondrial 0.23 PVRL1Poliovirus receptor-related protein 1 0.23 SLC46A2 Thymic stromalcotransporter homolog 0.23 ADORA3 Adenosine receptor A3 0.23 GPR125Probable G-protein coupled receptor 125 0.23 CD22 B-cell receptor CD220.22 FZD3 Frizzled-3 0.22 LPAR5 Lysophosphatidic acid receptor 5 0.22TMEM8B Transmembrane protein 8B 0.22 PLXNA1 Plexin-A1 0.22 NPFFR1Neuropeptide FF receptor 1 0.22 SEZ6L2 Seizure 6-like protein 2 0.22LRRTM2 Leucine-rich repeat transmembrane neuronal protein 2 0.22SLC16A11 Monocarboxylate transporter 11 0.22 GRIK5 Glutamate receptorionotropic, kainate 5 0.22 SYT6 Synaptotagmin-6 0.22 TMEM102Transmembrane protein 102 0.22 OR8B8 Olfactory receptor 8B8 0.22 GJB1Gap junction beta-1 protein 0.22 GRM6 Metabotropic glutamate receptor 60.22 C20orf54 Solute carrier family 52, riboflavin transporter, member 30.22 OR52D1 Olfactory receptor 52D1 0.22 SLC46A1 Proton-coupled folatetransporter 0.22 DSC2 Desmocollin-2 0.22 FAT1 Protocadherin Fat 1,nuclear form 0.22 GCGR Glucagon receptor 0.22 POP1 Blood vesselepicardial substance 0.22 CXADR Coxsackievirus and adenovirus receptor0.22 ABCC6 Multidrug resistance-associated protein 6 0.22 GJA1 Gapjunction alpha-1 protein 0.22 CXCR5 C-X-C chemokine receptor type 5 0.21ABCB4 Multidrug resistance protein 3 0.21 CTLA4 Cytotoxic T-lymphocyteprotein 4 0.21 TRPV1 Transient receptor potential cation channelsubfamily V member 1 0.21 MRGPRX4 Mas-related G-protein coupled receptormember X4 0.21 SIGLEC6 Sialic acid-binding Ig-like lectin 6 0.21 IL9RInterleukin-9 receptor 0.21 CHRNB2 Neuronal acetylcholine receptorsubunit beta-2 0.21 PDGFRB Platelet-derived growth factor receptor beta0.21 TMPRSS11D Transmembrane protease serine 11D catalytic chain 0.21CDH24 Cadherin-24 0.21 PRRT2 Proline-rich transmembrane protein 2 0.21GALR3 Galanin receptor type 3 0.21 OR51I1 Olfactory receptor 51I1 0.21PTPRU Receptor-type tyrosine-protein phosphatase U 0.21 LPAR4Lysophosphatidic acid receptor 4 0.21 ZNRF3 E3 ubiquitin-protein ligaseZNRF3 0.21 P2RY6 P2Y purinoceptor 6 0.21 AGTR1 Type-1 angiotensin IIreceptor 0.21 GPR182 G-protein coupled receptor 182 0.21 PODXLPodocalyxin 0.21 BDKRB1 B1 bradykinin receptor 0.21 DCHS1Protocadherin-16 0.21 GRIN3B Glutamate receptor ionotropic, NMDA 3B 0.21PTGDR Prostaglandin D2 receptor 0.21 PVRL4 Processed poliovirusreceptor-related protein 4 0.21 GPR77 C5a anaphylatoxin chemotacticreceptor 2 0.21 PARM1 Prostate androgen-regulated mucin-like protein 10.21 OR10H1 Olfactory receptor 10H1 0.21 OR10D3 Putative olfactoryreceptor 10D3 0.21 TNFSF14 Tumor necrosis factor ligand superfamilymember 14, soluble form 0.21 FCRL5 Fc receptor-like protein 5 0.2 RNF43E3 ubiquitin-protein ligase RNF43 0.2 AMIGO1 Amphoterin-induced protein1 0.2 OR1F1 Olfactory receptor 1F1 0.2 SLCO4A1 Solute carrier organicanion transporter family member 4A1 0.2 TTYH2 Protein tweety homolog 20.2 GABRR2 Gamma-aminobutyric acid receptor subunit rho-2 0.2 GJD3 Gapjunction delta-3 protein 0.2 GRID1 Glutamate receptor ionotropic,delta-1 0.2 CLDN1 Claudin-1 0.2 SLC6A13 Sodium- and chloride-dependentGABA transporter 2 0.2 SLC30A8 Zinc transporter 8 0.2 KIR2DL3 Killercell immunoglobulin-like receptor 2DL3 0.2 GPR78 G-protein coupledreceptor 78 0.2 UPK2 Uroplakin-2 0.2 CLDN14 Claudin-14 0.2 EDAEctodysplasin-A, secreted form 0.2 PTGER1 Prostaglandin E2 receptor EP1subtype 0.2 TRPV5 Transient receptor potential cation channel subfamilyV member 5 0.2 PRIMA1 Proline-rich membrane anchor 1 0.2 GJA9 Gapjunction alpha-9 protein 0.2 SLC7A3 Cationic amino acid transporter 30.2 SSTR2 Somatostatin receptor type 2 0.2 CD1A T-cell surfaceglycoprotein CD1a 0.2 SLC7A8 Large neutral amino acids transporter smallsubunit 2 0.2 CLIC6 Chloride intracellular channel protein 6 0.2 EPHA8Ephrin type-A receptor 8 0.2 SLC20A2 Sodium-dependent phosphatetransporter 2 0.2 SCNN1A Amiloride-sensitive sodium channel subunitalpha 0.2 OR51B6 Olfactory receptor 51B6 0.2 OR14J1 Olfactory receptor14J1 0.2 OR10C1 Olfactory receptor 10C1 0.2 OPRL1 Nociceptin receptor0.2 CCR9 C-C chemokine receptor type 9 0.2 JPH4 Junctophilin-4 0.2 HTR1E5-hydroxytryptamine receptor 1E 0.2 MC3R Melanocortin receptor 3 0.2CD163L1 Scavenger receptor cysteine-rich type 1 protein M160 0.2 SEZ6Seizure protein 6 homolog 0.2 PRSS8 Prostasin heavy chain 0.2 CDH26Cadherin-like protein 26 0.2 ODZ1 Teneurin C-terminal-associated peptide0.2 FGFR3 Fibroblast growth factor receptor 3 0.2

Example 1—Knock Out (Ko) on Cd38 Gene & Expression of Anti-Cd38 Car

Presentation of the CD38 Target—

cyclic ADP ribose hydrolase

CD38 is a glycoprotein found on the surface of many immune cells,including multiple myeloma (MM) cells that express a high level of CD38in a large majority of patients. CD38 is a validated target for MM asmany studies have shown efficient killing of CD38+MM cells from patientsand CD38+MM cell lines using anti-CD38 mAbs by CDC and ADCC (Ellis, J.H. K. et al, Journal of Immunology, 1995, 155 (2), 925-937). Daratumumabis a therapeutic human CD38 monoclonal antibody which induces killing ofmultiple myeloma and other hematological tumors (De Weers, M. et al, JImmunol 2011 186:1840-1848). In some studies, it has been shown thatCD38 is also highly expressed by activated T cells (Sandoval-Montes C Jet a, 2005, Leukoc Biol. 77(4):513-21).

Expression of CD38 by T-cells

The CD38 expression by T cells after CD3/CD28 beads and IL-2 stimulationwas analyzed by FACS every 3-4 days during 17 days. It was observed thatmore than 90% T cells express between day 6 and day 17 after activation(FIG. 10B).

Thus, in order to avoid killing of activated T cells by anti-CD38 CAR+ Tcells CD38 surface expression in T cells needs to be prevented. This maybe accomplished by the inactivation of the CD38 gene usingTALE-nucleases. TALEN is a trademark owned by the applicant (Cellectis,8 rue de la Croix Jarry, 75013 PARIS) designating customized format ofTAL nucleases.

Strategy for the CD38 Knock-Out (KO)

Heterodimeric TALE-nuclease targeting two 17-pb long sequences separatedby a 13-pb spacer within the CD38 gene were designed and produced. Eachhalf target is recognized by repeats of the half TALE-nucleases listedin the following Table 15 and FIG. 10A.

The repeats sequence of the left TALEN for the CD38ex1_T2 target wasNN-NI-NN-NN-NG-NN-NN-NN-NG-NG-NN-NN-HD-NN-NI-NG, and the one for theright TALEN was NN-NG-HD-HD-HD-HD-NN-HD-N I-NN-NG-NN-HD-HD-HD-NG.

TABLE 15Sequences of the tested CD38 target and TALENs for inactivation of the CD38 antigenTALEN Name L/R SEQ ID # Nucleic acid sequence or polypeptide sequenceCD38 N/A 1 TGAGGTGGGTTGGCGAC taaggcgcaccgg TGGGCACTGCGGGGACA targetCD38ex1_ L 2 MGDPKKKRKVIDYPYDVPDYAIDIADLRTLGYSQQQQEKIKPKVRSTV T2-AQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHE L1AIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTA TALENVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD CD38ex1_ R 3MGDPKKKRKVIDKETAAAKFERQHMDSIDIADLRTLGYSQQQQEKIKP T2-KVRSIVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAAL R1PEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAK TALENRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD

Each TALE-nuclease construct was subcloned using restriction enzymedigestion in a mammalian expression vector under the control of the T7promoter. mRNA encoding TALE-nuclease cleaving CD38 were synthesizedfrom plasmids carrying the coding sequence downstream from the T7promoter.

Purified T cells activated during 72 hours with anti CD3/CD28 coatedbeads and recombinant IL-2 were transfected by electroporation(Cytopulse) with each of the 2 mRNAs (10 μg each) encoding both halfCD38ex1_T2 TALE-nucleases. To investigate, the CD38 KO, the percentageof CD38 negative T cells was assessed by flow cytometry at day 3, 6, 10and 13 after TALEN mRNA transfection. It was observed that 15% oftransfected T cells were CD38 deficient (FIG. 10 C) and this deficiencywas stable during 13 days after transfection.

In addition two alternative TALE-nucleases targeting the CD38 gene havebeen designed. Each half target is recognized by repeats of the halfTALE-nucleases listed in the following Table 16 and FIG. 10A. Therepeats sequence of the left TALEN for the CD38ex1_T4 target wasNG-NN-HD-NN-NI-NN-NG-NG-HD-NI-NN-HD-HD-HD-NN-NN-NG, and the one for theright TALEN was NG-NN-HD-NG-NN-HD-HD-NN-NN-HD-NG-HD-NG-HD-NG-NI. Therepeats sequence of the left TALEN for the CD38ex1_T5 target wasNG-NN-NI-NG-HD-HD-NG-HD-NN-NG-HD-NN-NG-NN-NN-NG, and the one for theright TALEN was HD-NN-NI-NN-NN-NG-NN-NN-HD-NN-HD-HD-NI-NN-HD-NI.

TABLE 16Sequences of two other CD38 targets and the corresponding TALENs for theirinactivation TALEN Name L/R SEQ ID #Nucleic acid sequence or repeats sequence CD38ex1_ N/A 4TGCGAGTTCAGCCCGGtgtccggggacaaacccTGCTGCCGGCTCTCTA T4 target CD38ex1_ L 5MGDPKKKRKVIDYPYDVPDYAIDIADLRTLGYSQQQQEKIKPKVRSTVA T4-LQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEA TALENIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEV RRKFNNGEINFAADCD38ex1_ R 6 MGDPKKKRKVIDKETAAAKFERQHMDSIDIADLRTLGYSQQQQEKIKP T4-RKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAAL TALENPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGIlYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD CD38ex1_ N/A 7TGATCCTCGTCGTGGTgctcgcggtggtcgtccCGAGGTGGCGCCAGCA T5 target CD38ex1_ L 8MGDPKKKRKVIDYPYDVPDYAIDIADLRTLGYSQQQQEKIKPKVRSTVA T5-LQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEA TALENIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD CD38ex1_ R 9MGDPKKKRKVIDKETAAAKFERQHMDSIDIADLRTLGYSQQQQEKIKP T5-RKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAAL TALENPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNIGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD

Strategy for the Expression of the CAR Anti-CD38

Structure and Composition of CARs Anti-CD38

In Table 17 are presented VH and VL chain of scFv anti-CD38. SEQ IDNO:10-11 correspond to the humanized anti-CD38 antibody daratumumab(Genmab) and SEQ ID NO: 12-13 to the MOR202 (or MOR03087) such asdescribed in the U.S. Pat. No. 8,263,746B patent.

SEQ ID NO:14-20 and SEQ ID NO:21-26 correspond to the CDR sequence forrespectively the VH chain (HCDR) and the VL chain (LCDR) such asdescribed in the WO 2008/047242 application.

TABLE 17Sequences of VH and VL chains of the scFv anti-CD38 antibodies daratumumab,MOR202 and of specific CDRs for VH and VL chains. VH or VL Name chainSEQ ID # Polypeptide or nucleic acid sequence Daratumumab VH 10EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK VL 11EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MOR202 (or VH 12QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPG MOR03087)KGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS VL 13DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQ TYTGGASLVFGGGTKLTVLGQHCDR1-1 VH 14 GFTFSSYYMN HCDR1-2 VH 15 SYYMN HCDR2 VH 16GISGDPSNTYYADSVKG HCDR3 VH 17 DLPLVYTGFAY HCDR4 VH 18 DYWMQ HCDR5 VH 19TIYPGDGDTGYAQKFK HCDR6 VH 20 GDYYGSNSLDY LCDR1 VL 21 SGDNLRHYYVY LCDR2VL 22 GDSKRPS LCDR3 VL 23 QTYTGGASL LCDR4 VL 24 KASQDVSTVVA LCDR5 VL 25SASYRYI LCDR6 VL 26 QQHSPPYT

For the daratumumbab scFv 3 different CARs constructs (GMB005-V1&V2&V3)have been designed such as presented in FIG. 11A and their sequencedisplayed in the following Table 18. All three constructs share the samecomponents, in terms of signal peptide (CD8a), GS linker (between thescFv VH and VL chains), transmembrane domain (TM), 4-1BB costimulatorydomain, and CD3 activation domain (sequences displayed in the followingTable 18). Their differences come from the choice of the hinge (Table18):

V1: FcRIIa hinge

-   -   V2: CD8a hinge    -   V3: IgG1 hinge

TABLE 18Polypeptide sequence of the 3 different structures of scFv daratumumab-basedanti-CD38 CARs and of the individual components used Name of CARSEQ ID # CD8α-Signal 27 MALPVTALLLPLALLLHAARP peptide (SP) GSlinker 28GGGGSGGGGSGGGGS FCRIIα hinge 29 GLAVSTISSFFPPGYQ CD8α hinge 30TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCIgG1 hinge 31EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK TM domain 32IYIWAPLAGTCGVLLLSLVITLYC 4-1 BB co- 33KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL stimulatory domain CD3ζ 34RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE activationGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR domainGMB005-V1 35 PLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPG CARKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDAGMB005-V2 36 PLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPG CARKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA GMB005-V3 37PLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPG CARKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDA

Screening

CD38 TALENs will be transfected at day 4 after activation. 3 days afterthe CD38 deficient cells will be sorted by negative selection andtransfected 3 days after with anti-CD38 CAR mRNAs. The CAR moleculesgenerated will then be screened for expression and degranulationactivity toward target cell lines expression CD38 upon CAR mRNAtransient transfection. Target cell lines expressing differentexpression levels of CD38 (FIG. 11B) will be used for activity testing:

-   -   U266 CD38+ and U266 CD38-obtained by magnetic separation using        anti-CD38 microbeads    -   L363, a multiple myeloma cell line expressing intermediate        levels of CD38    -   Daudi, a cell line derived from Burkitt lymphoma expressing high        levels of CD38    -   K562, a cell line CD38 negative cell line derived from chronic        myelogenous leukemia.

This first screening will be followed by a second screening step inwhich a number of selected candidates will be tested for their abilityto induce degranulation, IFNγ release and specific cytotoxic activitytowards the selected target cell lines. Candidate selection will then benarrowed and some candidates selected for lentivirus vector productionand CAR activity will be assessed in CD38 KO T-cells stably expressingthe CARs.

Example 2 Activity of Anti-CS1 Car in the Context of CS1 Ko

Presentation of CS1 Target

Multiple myeloma (MM) is a B-cell malignancy characterized by theaberrant clonal expansion of plasma cells (PCs) within the bone marrow,with an estimated 21,700 new cases and 10,710 deaths from MM identifiedin the United States in 2012 (Siegel R, et al. Cancer J Clin 201262:10-29). In 2013, it has been estimated that 22,350 individuals willbe newly diagnosed with MM in the United States and 10,710 people willdie from it, accounting for 20% of the deaths from all hematologicmalignancies. Despite the use of proteasome inhibitors andimmune-modulating drugs, which have improved overall survival (PalumboA, et al. Leukemia 2009 23:449-456), MM remains an incurable malignancy(Podar K, et al. Leukemia 2009 23:10-24) for which novel therapeuticapproaches are urgently needed.

The cell surface glycoprotein CS1 (also referred in the literature asSLAMF7, CD319 or CRACC-NCBI Reference Sequence: NP_067004.3) is highlyand ubiquitously expressed on the surface of myeloma cells (Hsi E D, etal. Clin Cancer Res 2008 14:2775-84). CS1 is expressed at very lowlevels in the majority of immune effector cells, including naturalkiller (NK) cells, some subsets of T cells, and normal B cells, and isalmost undetectable on myeloid cells (Hsi E D, et al. Clin Cancer Res2008 14:2775-84). Notably, CS1 is negligibly expressed in humanhematopoietic stem cells (Hsi E D, et al. Clin Cancer Res 200814:2775-84), which can be used for stem cell transplantation to treathematologic malignancies, including MM. The functions of CS1 in MMremain incompletely understood, and it has been documented that CS1 mayplay a role in myeloma cell adhesion, clonogenic growth, andtumorigenicity (Benson D M Jr, et al. J Clin Oncol 2012 30:2013-5; Tai YT, et al. Blood 2009 113:4309-18).

Structure of the CAR Anti-CS1

The same structures V1, V2 and V3 are designed such as in the Example 1for the anti-CD38 antigen target single-chain CAR, with the samecomponents in terms of hinge, transmembrane domain, co-activation andtransduction domains (such as depicted in the FIG. 11A and sequencesshown in Table 18).

In Table 19 are presented the VH and VL chains of scFv anti-CS1. SEQ IDNO:38-40-42-44-46 and SEQ ID NO:39-41-43-45-47 correspond torespectively the VH chain and the VL chain of the murine scFv Luc63,Luc90, Luc34, LucX1 and LucX2.

In Table 20 are presented anti-CS1 CARs with the above scFv; these CARsbeing based on the versions V1, V2 and V3 of FIG. 11A, whereinrespectively the short FcERγ hinge, the medium hinge CD8a hinge and thelong IgG1 hinge are used. The underlined parts correspond to the scFv VHand VL chains bound by a linker.

TABLE 19 Sequences of VH and VL chains of the scFv anti-CS1 antibodiesVH or VL SEQ ID Name chain NO: Polypeptide sequence Luc63 VH 38EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARPDGN YWYFDVWGAGTTVTVSSVL 39 DIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGG GTKLEIK Luc90 VH 40QVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSS VL 41DIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYYCQQHYSTPLTFGAG TKLELK Luc34 VH 42QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAYWGQGTLVTVSA VL 43DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGG TKLEIK LucX1 VH 44QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSS VL 45ETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGGGTKL EIK LucX2 VH 46QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSS VL 47DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPPYTFGG GTKLEIK

TABLE 20Polypeptide sequence of anti-CS1 CARs based on the V1, V2 and V3 versions in FIG. 11AName of CAR SEQ ID # Polypeptide sequence Luc63- 48MALPVTALLLPLALLLHAARPEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLV1 CAREWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARPDGNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK GLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Luc63- 49MALPVTALLLPLALLLHAARPEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLV2 CAREWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARPDGNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Luc63- 50MALPVTALLLPLALLLHAARPEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLV3 CAREWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARPDGNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVGIAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Luc90- 51MALPVTALLLPLALLLHAARPQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQ V1 CARGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Luc90- 52MALPVTALLLPLALLLHAARPQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQ V2 CARGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYYCQQHYSTPLTFGAGTKLEL KTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Luc90- 53MALPVTALLLPLALLLHAARPQVQLQQPGAELVRPGASVKLSCKASGYSFTTYWMNWVKQRPGQ V3 CARGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARSTMIATRAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSQKSMSTSVGDRVSITCKASQDVITGVAWYQQKPGQSPKWYSASYRYTGVPDRFTGSGSGTDFTFTISNVQAEDLAVYYCQQHYSTPLTFGAGTKLEL KEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Luc34- 54MALPVTALLLPLALLLHAARPQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQG V1 CARLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPVVTFGGGTKLEIKGLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Luc34- 55MALPVTALLLPLALLLHAARPQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQG V2 CARLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPVVTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Luc34- 56MALPVTALLLPLALLLHAARPQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQG V3 CARLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPVVTFGGGTKLEIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLIVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR LucX1- 57MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQG V1 CARLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGGGTKLEIK GLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR LucX1- 58MALPVTALLLPLALLLHAARPQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQG V2 CARLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARGKVYYGSNPFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPVVTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR LucX1- 59MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQG V3 CARLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSETTVTQSPASLSMAIGEKVTIRCITSTDIDDDMNWYQQKPGEPPKLLISEGNTLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPLTFGGGTKLEIK EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR LucX2- 60MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQG V1 CARLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPPYTFGGGTKLEI KGLAVSTISSFFPPGYQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR LucX2- 61MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQG V2 CARLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPPYTFGGGTKLEI KTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR LucX2- 62MALPVTALLLPLALLLHAARPQVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGQG V3 CARLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSVDSAVYFCARSTMIATGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPPYTFGGGTKLEI KEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Strategy for CAR CS1+ and KO CS1 Engineering

CS1 is expressed at high levels in plasmacytoid cells from patients withMultiple Myeloma, making this an interesting target for CAR development.T-cells, especially the CD8 subset, express low levels of CS1, which isa drawback for T-cell CAR development, since they could be killed whenexpressing an anti-CS1 CAR.

In this example we assessed the activity of the Luc90-v2 CAR (sequenceshown in Table 20) in human T-cells that were either mock transfected,or transfected with a TALEN targeting the CS1 (SLAMF7) gene, to see ifthe CAR activity was enhanced when the CS1 gene was disrupted in CAR+T-cells. The course of the experiment is shown in the FIG. 12.

T-cells were purified from buffy-coat samples and activated usingCD3/CD28-coated beads. Cells were co-transfected 72 h after activationwith 10 μg of mRNA encoding the T01_left TAL and 10 μg of the mRNAencoding the T01_right TAL. Sequences of the TALs are shown in thefollowing Table 21 and the plasmid constructs (T01, T02 and T03) withthe TAL repeats shown in FIG. 13.

FIG. 14 shows the target location for the TALs T01, T02 and T03 withinthe CS1 (SLAMF7) gene: T01 and T02 target the exon 1 (FIG. 14A), whereasT03 targets the exon 2 (FIG. 14B).

TABLE 21 Sequences of the CS1 target and TALENs for its inactivationTALEN Name L/R SEQ ID # Nucleic acid sequence Target of T01 63TGACTTCCAGAGAGCAATATGGCTGGTTCCCCAACATGCCTC ACCCTCA L 64TGACTTCCAGAGAGCAA R 65 AACATGCCTCACCCTCA Target of T02 66TTCCAGAGAGCAATATGGCTGGTTCCCCAACATGCCTCACCC TCATCTA L 67TTCCAGAGAGCAATATG R 68 TGCCTCACCCTCATCTA Target of T03 69TTGACTCTATTGTCTGGACCTTCAACACAACCCCTCTTGTCAC CATACA L 70TTGACTCTATTGTCTGG R 71 CCTCTTGTCACCATACA

3 days after TALEn transfection, cells were transduced with arecombinant lentiviral vector driving expression of the L90-v2 CAR offan EF1a promoter. The lentiviral vector is built in a way that CARexpression is coupled with BFP expression (Blue Fluorescent Protein)through a ribosomal skip peptide. The L90-v2 CAR is constituted by anextracellular binding domain recognizing the CS1 target (scFv L90)followed by hinge and transmembrane regions derived from the hCD8αprotein. The intracellular portion of the molecule contains a416B-derived costimulatory domain, followed by the CD3γ signaling domain(sequences displayed in previous Table 18-19-20 for individualcomponents, scFv and CAR sequences respectively).

Transduction efficiency was assessed 6 days after transduction by flowcytometry, by following BFP expression. Cells were also stained withanti-CD8 and anti-CS1 antibodies.

Results

CAR CS1+ Expression

The results from FIG. 16 show that the transduction efficiencies arehigher in mock transfected cells than in cells that have beentransfected with TALEn targeting the CS1 gene. This is probably due tospecific cell killing of non-transduced CS1-expressing T-cells, whilethis population is not affected when the cells no longer express CS1 asa consequence of TALEN-driven gene disruption.

No significant differences in CS1 levels are observed at this timepointbetween TALEN or mock transfected cells (negative control-transfectionwithout plasmid), since CS1 levels decrease over time after initialactivation of T-cells. On the other hand, a significant decrease in the% of CD8+ cells is observed in mock transfected CAR expressing cellscompared to TALEN transfected CAR+ cells, indicating that a highproportion of CD8+ cells has been eliminated by the CAR+ T-cells.

Cytotoxic Activity Assessment

The cytotoxic activity of these cells was evaluated 8 days after CARtransduction, by co-culturing the same amount of T-cells either with acell line expressing CS1 (L363 cells) or a negative control cell linelacking expression of CS1 (MOLM13). The viability of the target celllines was measured by flow cytometry 4 h after starting cellco-cultures. The results shown in FIG. 15A show reduced cell viabilityof CS1(+) cells when they were co-cultured with CAR+ T-cells, while noimpact on CS1(−) cell viability was observed. The specific cell lysiswas calculated using the flow cytometry data, and it was 2-times higherwhen T-cells have been transfected with TALEn targeting the CS1 geneprior to CAR transduction (FIG. 15B). It should be considered that theimpact might be even higher, since the amount of CAR+ T-cells present inthe co-cultures is higher when the cells were mock transfected (see flowcytometry data from FIG. 16). The results from the experiment are thefollowing:

-   -   for the Mock/NTD sample, the % of BFP+ cells is 0.1% and the        amount of CD8+ cells is 53.9%;    -   for the TALEn/NTD sample, the % of BFP+ cells is 0.2% and the        amount of CD8+ cells is 49.5%;    -   for the Mock/L90-2 sample, the % of BFP+ cells is 94% and the        amount of CD8+ cells is 1.8%;    -   for the TALEn/L90-2 sample, the % of BFP+ cells is 61% and the        amount of CD8+ cells is 8.3%.    -   Transduction efficiencies are higher in mock transfected cells        than in cells that have been transfected with TALEn targeting        the CS1 gene (NTD: not transduced).

Reactivation after Transduction

In order to confirm that the CS1 gene has been disrupted in TALEntransfected T-cells, the different samples were reactivated withCD3/CD28 beads at D11 after transduction. 72 h after reactivation cellswere stained with anti-CD8 and anti-CS1 antibodies and expressionanalyzed by flow cytometry.

FIG. 17 shows the transduction efficiencies and CD8/CS1 expressionlevels in each sample. As shown in the lower panel, an increase in CS1levels upon re-activation is observed in mock transfected cells, while alow amount of cells are able to express CS1 in the TALEn transfectedpopulations.

The results from the experiment are the following:

-   -   for the Mock/NTD sample, the % of BFP+ cells is 0.01%, CS1 is        expressed in 65.2% of cells, and the amount of CD8+ cells is        80.7%;    -   for the TALEn/NTD sample, the % of BFP+ cells is 0.2%, the CS1        is expressed in 9.7% of cells and the amount of CD8+ cells is        78.8%;    -   for the Mock/L90-2 sample, the % of BFP+ cells is 94%, the CS1        is expressed in 37.5% of cells and the amount of CD8+ cells is        16%.    -   for the TALEn/L90-2 sample, the BFP intensity is 61%, the CS1        expression is 8.5% and the CD8 expression is 68.5%.

An increase in CS1 levels upon re-activation is observed in mocktransfected cells, while a low amount of cells are able to express CS1in the TALEn transfected populations.

Altogether, these results indicate that the CS1 gene is disrupted inTALEn transfected T-cells, and that this enhances the cytotoxic activityof anti-CS1 CAR+ cells, mainly by preserving the cytotoxic CD8+ T-cells.

Example 3: Cd70 Target

Presentation of CD70 Target

The CD70 is a cytokine that binds to CD27 and is part of the TNF family(Goodwin R. G. et al, 1993, Cell 73:447-456). This protein has a role inadaptive T cell responses, induces the proliferation of costimulatedT-cells and enhances the generation of cytolytic T-cells. Its accessionnumber is P32970 (Uniprot). Some studies such as in Schürch, C. et al.(J. Clin. Invest., 2012; doi:10.1172/JC145977) suggest that blockingCD27-CD70 interactions could help treat chronic myelogenous leukemia(CML).

Strategy for CD70 KO

The same strategy for the KO of CD70 gene will be performed such as inExample 1 and Example 2. Heterodimeric TALE-nuclease targeting two 49-pblong sequences separated by a 15 pb spacer within the CD70 gene and oneTALE-nuclease targeting a 57-pb long sequence separated by a 23 pbspacer were designed and produced. Each half target is recognized byrepeats of the half TALE-nucleases listed in the following Table 22.

TABLE 22 Sequences of the CD70 target and TALENs for its inactivationTALEN Name L/R SEQ ID # Nucleic acid sequence Target 1 72TGGTCTTTTCTTCCAGTgggacgtagctgagcTGCAGCTGAATCACACA TALEN 1 L 73TGGTCTTTTCTTCCAGT R 74 TGCAGCTGAATCACACA Target 2 75TGGTGATCTGCCTCGTGgtgtgcatccagcgcTTCGCACAGGCTCAGCA TALEN 2 L 76TGGTGATCTGCCTCGTG R 77 TTCGCACAGGCTCAGCA Target 3 78TGCGGGCTGCTTTGGTCccattggtcgcgggcttggtgatCTGCCTCGTGGTG TALEN 3 TGCA L 79TGCGGGCTGCTTTGGTC R 80 CTGCCTCGTGGTGTGCA

Strategy for the Expression of Anti-CD70 CAR

The same strategy for expressing a CAR anti-CD70 will be performed suchas in Example 1 and in Example 2.

The same structures V1, V2 and V3 are designed such as in the Example1-2 with the same components in terms of signal peptide, linker betweenthe VH and VL chains, transmembrane domain, co-activation andtransduction domains (general architectures shown in FIG. 11A, andsequences for individual components shown in Table 18). Only the hingediffers between the 3 versions V1, V2 and V3, wherein respectively theshort FcERγ hinge, the medium hinge CD8a hinge and the long IgG1 hingeare used.

In Table 23 are presented VH and VL chain of scFv anti-CD70. SEQ IDNO:81-82, 85-86, 89-90 and SEQ ID NO:83-84,87-88,91-92 correspond torespectively the VH chain and the VL chain of the scFv Ab4, Ab8 fromAMGEN and 1F6 from Seattle Genetics.

In Table 24 are presented the anti-CD70 CARs with the above scFv; theseCARs being based on the versions V1, V2 and V3 according to FIG. 11A,wherein respectively a short FcEγ hinge, a medium hinge CD8 and a longIgG1 hinge are used.

TABLE 23Polynucleotide and nucleic acid sequences of VH and VL chains for the scFv anti-CD70 Ab4, Ab8 and 1F6 antibodies VH or VL Name chain SEQ ID #Polypeptide and nucleic acid sequence Ab4 VH 81QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGIHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSGYDSGFDYWGQGTLVTVSS 82caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtaactatggcatacactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagatggaggatatagtggctacgattcggggtttgactactggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatccgtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga VL 83DIVMTQSPLSLPVTPGEPASISCRSSQSLLNSNGYNYLDWYLQKPGQSPQFLIYLGSYRASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCIQTLQ TPFTFGPGTKVDIK 84Gatattgtgatgactcagtctccactctccctgcccgtcacccctggagagccggcctccatctcctgcaggtctagtcagagcctcctgaatagtaatggatacaactatttggattggtacctgcagaagccagggcagtctccacagttcctgatctatttgggttcttatcgggcctccggggtccctgacaggttcagtggcagtggatcaggcacagattttacactgagaatcagcagagtggaggctgaggatgttggggtttattactgtatacaaactctacaaactccattcactttcggccctgggaccaaagtggatatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagtcctca53ggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa Ab8 VH 85QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSDKYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGIAGARYVYFDYWGQGTLVTVSS 86caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtgataaatactttgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagatgggatagcaggagctcgctacgtctactttgactactggggccagggaaccctggtcaccgtctcctcagctagcaccaagggcccatccgtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccct VL 87DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQGGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYYNYPFTF GPGTTVDIK 88gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggcattagcaattatttagcctggtttcagcagaaaccagggaaagcccctaagtccctgatctatgctgcatccagtttgcaaggtggggtcccatcaaagttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacaatattataattacccattcactttcggccctgggaccacagtggatatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag 1F6 VH 89QIQLVQSGPEVKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGINTYTGEPTYADAFKGRFAFSLETSASTAYLQINNLKNEDTATYF CARDYGDYGMDYWGQGTSVTVSS90 atggcttgggtgtggaccttgctattcctgatggcagctgcccaaagtgcccaagcacagatccagttggtgcagtctggacctgaggtgaagaagcctggagagacagtcaagatctcctgcaaggcttctgggtataccttcacaaactatggaatgaactgggtgaagcaggctccaggaaagggtttaaagtggatgggctggataaacacctacactggagagccaacatatgctgatgccttcaagggacggtttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgtgcaagagactacggcgactatggtatggactactggggtcaaggaacctcagtcaccgtctcctca VL 91DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREV PWTFGGGTKLEIKRatggagacagacacactcctgttatgggtactgctgctctgggttccaggttccactggtgacattg 92tgctgacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggctatagttttatgcactggtatcaacagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggaggttccgtggacgttcggtggaggcaccaagctggaaatcaaacgg

TABLE 24Polypeptide sequences of anti-CD70 CARs based on the V1, V2 and V3 versionsaccording to FIG. 11A Name SEQ ID of CAR NO: Polypeptide sequence Ab4-V193 MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGIHWVR CARQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSGYDSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLNSNGYNYLDWYLQKPGQSPQFLIYLGSYRASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCIQTLQTPFTFGPGTKVDIKGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Ab4-V2 94MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGIHWVR CARQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSGYDSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLNSNGYNYLDWYLQKPGQSPQFLIYLGSYRASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCIQTLQTPFTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Ab4-V3 95MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGIHWVR CARQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGYSGYDSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLNSNGYNYLDWYLQKPGQSPQFLIYLGSYRASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCIQTLQTPFTFGPGTKVDIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPRAb8-V1 96 MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWV CARRQAPGKGLEWVAVIWYDGSDKYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGIAGARYVYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQGGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYYNYPFTFGPGTTVDIKGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Ab8-V2 97MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWV CARRQAPGKGLEWVAVIWYDGSDKYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGIAGARYVYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQGGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYYNYPFTFGPGTTVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPRAb8-V3 98 MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWV CARRQAPGKGLEWVAVIWYDGSDKYFADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGIAGARYVYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQGGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYYNYPFTFGPGTTVDIKEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR1F6 V1 99 MALPVTALLLPLALLLHAARPQIQLVQSGPEVKKPGETVKISCKASGYTFTNYGMNWVKCAR QAPGKGLKWMGINTYTGEPTYADAFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYGDYGMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREVPWTFGGGTKLEIKRGLAVSTISSFFPPGYQIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 1F6 V2 100MALPVTALLLPLALLLHAARPQIQLVQSGPEVKKPGETVKISCKASGYTFTNYGMNWVK CARQAPGKGLKWMGINTYTGEPTYADAFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYGDYGMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREVPWTFGGGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR1F6 V3 101 MALPVTALLLPLALLLHAARPQIQLVQSGPEVKKPGETVKISCKASGYTFTNYGMNWVKCAR QAPGKGLKWMGINTYTGEPTYADAFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYGDYGMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSREVPWTFGGGTKLEIKREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR

REFERENCES

-   Bardenheuer, W., K. Lehmberg, et al. (2005). “Resistance to    cytarabine and gemcitabine and in vitro selection of transduced    cells after retroviral expression of cytidine deaminase in human    hematopoietic progenitor cells.” Leukemia 19(12): 2281-8.-   Betts, M. R., J. M. Brenchley, et al. (2003). “Sensitive and viable    identification of antigen-specific CD8+ T cells by a flow cytometric    assay for degranulation.” J Immunol Methods 281(1-2): 65-78.-   Boch, J., H. Scholze, et al. (2009). “Breaking the code of DNA    binding specificity of TAL-type III effectors.” Science 326(5959):    1509-12.-   Brewin, J., C. Mancao, et al. (2009). “Generation of EBV-specific    cytotoxic T cells that are resistant to calcineurin inhibitors for    the treatment of posttransplantation lymphoproliferative disease.”    Blood 114(23): 4792-803.-   Cambier, J. C. (1995) “Antigen and Fc Receptor Signaling: The    Awesome Power of the Immunoreceptor Tyrosine-I Based Activation    Motif (ITAM)” The Journal of Immunology 155 (7) 3281-3285.-   Cong, L., F. A. Ran, et al. (2013). “Multiplex genome engineering    using CRISPR/Cas systems.” Science 339(6121): 819-23.-   Critchlow, S. E. and S. P. Jackson (1998). “DNA end-joining: from    yeast to man.” Trends Biochem Sci 23(10): 394-8.-   Dalgaard, J. Z., A. J. Klar, et al. (1997). “Statistical modeling    and analysis of the LAGLIDADG family of site-specific endonucleases    and identification of an intein that encodes a site-specific    endonuclease of the HNH family.” Nucleic Acids Res 25(22): 4626-38.-   Deltcheva, E., K. Chylinski, et al. (2011). “CRISPR RNA maturation    by trans-encoded small RNA and host factor RNase III.” Nature    471(7340): 602-7.-   Garneau, J. E., M. E. Dupuis, et al. (2010). “The CRISPR/Cas    bacterial immune system cleaves bacteriophage and plasmid DNA.”    Nature 468(7320): 67-71.-   Gasiunas, G., R. Barrangou, et al. (2012). “Cas9-crRNA    ribonucleoprotein complex mediates specific DNA cleavage for    adaptive immunity in bacteria.” Proc Natl Acad Sci USA 109(39):    E2579-86.-   Hacke, K., J. A. Treger, et al. (2013). “Genetic modification of    mouse bone marrow by lentiviral vector-mediated delivery of    hypoxanthine-Guanine phosphoribosyltransferase short hairpin RNA    confers chemoprotection against 6-thioguanine cytotoxicity.”    Transplant Proc 45(5): 2040-4.-   Jena, B., G. Dotti, et al. (2010). “Redirecting T-cell specificity    by introducing a tumor-specific chimeric antigen receptor.” Blood    116(7): 1035-44.-   Jinek, M., K. Chylinski, et al. (2012). “A programmable    dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.”    Science 337(6096): 816-21.-   Jonnalagadda, M., C. E. Brown, et al. (2013). “Engineering human T    cells for resistance to methotrexate and mycophenolate mofetil as an    in vivo cell selection strategy.” PLoS One 8(6): e65519.-   Kushman, M. E., S. L. Kabler, et al. (2007). “Expression of human    glutathione S-transferase P1 confers resistance to benzo[a]pyrene or    benzo[a]pyrene-7,8-dihydrodiol mutagenesis, macromolecular    alkylation and formation of stable N2-Gua-BPDE adducts in stably    transfected V79MZ cells co-expressing hCYP1A1.” Carcinogenesis    28(1): 207-14.-   Lackner, G., N. Moebius, et al. (2011). “Complete genome sequence of    Burkholderia rhizoxinica, an Endosymbiont of Rhizopus microsporus.”    J Bacteriol 193(3): 783-4.-   Ma, J. L., E. M. Kim, et al. (2003). “Yeast Mre11 and Rad1 proteins    define a Ku-independent mechanism to repair double-strand breaks    lacking overlapping end sequences.” Mol Cell Biol 23(23): 8820-8.-   Mak, A. N., P. Bradley, et al. (2012). “The crystal structure of TAL    effector PthXo1 bound to its DNA target.” Science 335(6069): 716-9.-   Mali, P., L. Yang, et al. (2013). “RNA-guided human genome    engineering via Cas9.” Science 339(6121): 823-6.-   Metzger, H. et al. (1986) “The Receptor with High Affinity for    Immunoglobulin E” Annual Review of Immunology. 4: 419-470-   Moscou, M. J. and A. J. Bogdanove (2009). “A simple cipher governs    DNA recognition by TAL effectors.” Science 326(5959): 1501.-   Nivens, M. C., T. Felder, et al. (2004). “Engineered resistance to    camptothecin and antifolates by retroviral coexpression of tyrosyl    DNA phosphodiesterase-I and thymidylate synthase.” Cancer Chemother    Pharmacol 53(2): 107-15.-   Park, T. S., S. A. Rosenberg, et al. (2011). “Treating cancer with    genetically engineered T cells.” Trends Biotechnol 29(11): 550-7.-   Sangiolo, D., M. Lesnikova, et al. (2007). “Lentiviral vector    conferring resistance to mycophenolate mofetil and sensitivity to    ganciclovir for in vivo T-cell selection.” Gene Ther 14(21):    1549-54.-   Schweitzer, B. I., A. P. Dicker, et al. (1990). “Dihydrofolate    reductase as a therapeutic target.” Faseb J 4(8): 2441-52.-   Sugimoto, Y., S. Tsukahara, et al. (2003). “Drug-selected    co-expression of P-glycoprotein and gp91 in vivo from an    MDR1-bicistronic retrovirus vector Ha-MDR-IRES-gp91.” J Gene Med    5(5): 366-76.-   Takebe, N., S. C. Zhao, et al. (2001). “Generation of dual    resistance to 4-hydroperoxycyclophosphamide and methotrexate by    retroviral transfer of the human aldehyde dehydrogenase class 1 gene    and a mutated dihydrofolate reductase gene.” Mol Ther 3(1): 88-96.-   Waldmann H. and Hale G. (2005) “CAMPATH: from concept to clinic”.    Phil. Trans. R. Soc. B 360: 1707-1711.-   Yam, P., M. Jensen, et al. (2006). “Ex vivo selection and expansion    of cells based on expression of a mutated inosine monophosphate    dehydrogenase 2 after HIV vector transduction: effects on    lymphocytes, monocytes, and CD34+ stem cells.” Mol Ther 14(2):    236-44.-   Zielske, S. P., J. S. Reese, et al. (2003). “In vivo selection of    MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells    without pretransplant irradiation conditioning.” J Clin Invest    112(10): 1561-70.

1) A method of preparing immune cells, preferably T-cells, forimmunotherapy against pathological cells comprising the step of: (c)Genetically inactivating or mutating a gene in a immune cell, which isinvolved in the expression or presentation of an antigen marker, saidantigen marker being present both on the surface of said immune cell andthe pathological cell; (d) Expressing into said immune cell a transgeneencoding a chimeric antigen receptor directed against said antigenmarker present at the surface of said pathological cell. 2) A methodaccording to claim 1, wherein said antigen marker is selected from onelisted in Table
 4. 3) A method according to claim 1, wherein saidantigen marker is CD38 or an immuno-reactive fragment thereof. 4) Amethod according to any one of claims 1 to 3, wherein said methodincludes a further step of activating and expanding the immune cells. 5)A method according to any one of claims 1 to 4, wherein said methodincludes a further step of purifying the resulting immune cells byexcluding the cells presenting said marker antigen at their surface. 6)A method according to any one of claims 1 to 5, wherein said methodincludes a previous step of procuring the immune cells from a donor 7) Amethod according to any one of claims 1 to 5, wherein said methodincludes a previous step of procuring the immune cells from a patientwho is affected by the development of said pathological cells. 8) Amethod according to any one of claims 1 to 7, wherein said immune cellis derived from a primary stem cell, iPS or hES cell. 9) A methodaccording to claim 8, wherein said immune cell is derived from iPS cellderived from said patient affected by the development of saidpathological cells. 10) A method according to any one of claims 1 to 9,wherein step a) is performed using a rare-cutting endonuclease. 11) Amethod according to claim 10, wherein step a) is performed using aTAL-nuclease. 12) A method according to claim 10, wherein step a) isperformed using a RNA-guided endonuclease 13) A method according toclaim 12, wherein the RNA-guided endonuclease is Cas9. 14) A methodaccording to claim 12, wherein RNA-guided endonuclease is split into atleast 2 polypeptides, one comprising RuvC and another comprising HNH.15) A method according to claim 10, wherein said endonuclease isexpressed from transfected mRNA. 16) A method according to any one ofclaims 1 to 15, wherein said method includes a further step ofinactivating a gene encoding a component of the T-cell receptor (TCR).17) A method according to claim 16, wherein said component of the T-cellreceptor is TCRα. 18) A method according to any one of claims 1 to 15,wherein said method includes a further step of inactivating a geneencoding a component of HLA. 19) A method according to any one of claims1 to 15, wherein said method includes a further step of inactivating agene encoding β2m. 20) A method according to any one of claims 1 to 15,wherein said method includes a further step of inactivating a geneencoding an immune checkpoint protein selected from CTLA4, PPP2CA,PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96,CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8,CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3,SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST,EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2and GUCY1B3. 21) A method according to claim 20, wherein said gene locusis involved into the expression of PD1 or CTLA-4 genes. 22) A methodaccording to any one of claims 1 to 15, wherein said method includes afurther step of inactivating a gene conferring sensitivity of the immunecells to chemotherapy or immunosuppressive drugs. 23) The methodaccording to claim 22, wherein said further gene encodes CD52. 24) Themethod according to claim 22, wherein said further gene ishypoxanthine-guanine phosphoribosyltransferase (HPRT). 25) The methodaccording to claim 22, wherein said further gene encodes aglucocorticoid receptor (GR). 26) The method according to claim 22,wherein said further gene is involved in the DCK regulatory pathway, inparticular DCK expression. 27) A method according to any one of claims 1to 26, wherein said immune cells in step a) are derived frominflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatoryT-lymphocytes or helper T-lymphocytes. 28) The method according to claim27, wherein said T-cells are derived from CD4+T-lymphocytes and/orCD8+T-lymphocytes. 29) A method according to any one of claims 1 to 28,wherein said transformed immune cells are expanded in-vitro. 30) Amethod according to any one of claims 1 to 28, wherein said transformedimmune cells are expanded in-vivo. 31) A method according to any one ofclaims 1 to 30, wherein said pathological cells are selected frommalignant cells or infected cells. 32) A method according to any one ofclaims 1 to 30, wherein said pathological cells are B-cells. 33) Amethod according to any one of claims 1 to 30, wherein said pathologicalcells are solid tumor cells. 34) A method according to any one of claims1 to 30, for preparing immune cells to be used as a medicament. 35) Amethod according to claim 31 for preparing immune cells for treating acancer, an immune disease or an infection in a patient in need thereof.36) A method according to claim 32 for treating lymphoma. 37) A methodaccording to claim 32 for treating leukemia. 38) A method according toclaim 34 for treating chronic lymphocytic leukemia (CLL). 39) Anengineered immune cell obtainable according to the method of any one ofclaims 1 to
 35. 40) An engineered immune cell according to claim 39resulting into the phenotype [CAR CD38]⁺[CD38]⁻ 41) An engineered immunecell according to claim 39 resulting into the phenotype [CARCD70]⁺[CD70]⁻ 42) An engineered immune cell according to claim 39resulting into the phenotype [CAR CS1]⁺[CS1]⁻ 43) A method for treatinga patient comprising: (a) Diagnosing said patient for the presence ofpathological cells presenting specific antigen markers in common withimmune cells; (b) preparing a population of engineered immune cellsaccording to claims 38 to 42 or according to the method of any one ofclaims 1 to 35; and (c) administrating said engineered immune cells tosaid patient diagnosed for said pathological cells.